US5166322A - Cysteine added variants of interleukin-3 and chemical modifications thereof - Google Patents

Cysteine added variants of interleukin-3 and chemical modifications thereof Download PDF

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US5166322A
US5166322A US07341990 US34199089A US5166322A US 5166322 A US5166322 A US 5166322A US 07341990 US07341990 US 07341990 US 34199089 A US34199089 A US 34199089A US 5166322 A US5166322 A US 5166322A
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cavs
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Gray Shaw
Geertruida Veldman
Joseph L. Wooters
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Genetics Institute LLC
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/5403IL-3
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/505Erythropoietin [EPO]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/53Colony-stimulating factor [CSF]
    • C07K14/535Granulocyte CSF; Granulocyte-macrophage CSF
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00Peptide or protein sequence
    • Y10S930/01Peptide or protein sequence
    • Y10S930/14Lymphokine; related peptides
    • Y10S930/141Interleukin

Abstract

Cysteine added variants ("CAVs") of interleukin-3 are provided having one or more cysteine residues substituted for selected naturally occurring amino acid residues, or inserted into the polypeptide sequence, and preferably being further modified by deletion of certain N-terminal amino acids. Such CAVs may be additionally modified by the coupling of sulfhydryl reactive compounds to the introduced cysteine residue(s) without loss of bioactivity to produce selected homogeneously modified IL-3 and improved pharmaceutical compositions containing the same.

Description

TECHNICAL FIELD

This invention relates generally to interleukin-3 (IL-3) polypeptides modified by the attachment of compounds having sulfhydryl reactive groups, improved methods for producing such modified IL-3 polypeptides and improved compositions containing them. More particularly, the invention relates particularly to modified IL-3, to which sulfhydryl reactive compounds, including polymers, may be attached at selected positions in the polypeptide that have been modified by the insertion of cysteine residues or the substitution of cysteine residues for other residues.

BACKGROUND

The desirability of modifying biologically active and therapeutically useful polypeptides with a variety of compounds, such as the hydrophilic polymer polyethylene glycol (PEG), to enhance their pharmacokinetic properties has been noted. See, e.g., the discussion of the art in this area of polypeptide modification in published PCT patent application WO87/00056, in U.S. Pat. No. 4,179,337, which discloses conjugating water soluble polypeptides such as enzymes and insulin to PEG or PPG, and in U.S. Pat. No. 4,766,106, which discloses conjugating ordinarily water insoluble beta-interferon, interleukin-2, or immunotoxins to PEG homopolymers or polyoxyethylated glycerol. Such modification can reduce adverse immune response to the polypeptide, increase the solubility for use in pharmaceutical preparations and maintain a desirable circulatory level of such polypeptide for therapeutic efficacy.

One problem not addressed by the art in this area involves the extent to which a polypeptide can be modified by attachment of compounds having reactive groups that will covalently bond to certain amino acid residues of the polypeptide For example, modification of a polypeptide with PEG or similar polymers, can result in random attachment of the polymer at the amino terminus of the polypeptide and/or at one or more lysine residues in the amino acid sequence of the protein Because more than one PEG group can attach to the polypeptide, the resultant composition may contain a heterogeneous mixture of "PEGylated" polypeptide; some polypeptides having only one PEGylated site, others having more than one PEGylated site. Such heterogeneity in composition is undesirable for pharmaceutical use. Furthermore, the non-specificity with regard to the site(s) of attachment of compounds such as PEG to the polypeptide can result in loss of biological efficacy of the polypeptide stemming from undesirable attachment to a polypeptide site required for biological activity.

Co-pending, commonly owned U.S. patent application Ser. No. 137,043 now U.S. Pat. No. 4,904,584 entitled SITE-SPECIFIC HOMOGENEOUS MODIFICATION OF POLYPEPTIDES addresses the foregoing by providing materials and methods for site specific covalent modification of polypeptides by lysine insertion, removal, and/or replacement. However, we have determined that the use of lysine as the attachment site for modification, for example, by PEGylation, may be disadvantageous in the case of IL-3 because not all modifications may result in biologically active compounds and because steps must be taken to prevent PEGylation at N-termini in cases where N-terminal PEGylation is not desired.

SUMMARY OF THE INVENTION

This invention provides materials and methods for site specific covalent modification of IL-3 polypeptides, preferably human IL-3 polypeptides, permitting the production of compositions comprising homogeneously cys modified IL-3s and pharmaceutical compositions containing the same. "Homogeneously cys modified" as the term is used herein means substantially consistently modified only at specific, inserted or substituted cysteine residues. A homogeneously modified IL-3 for example, includes an IL-3 composition which is substantially consistently modified at position 6 (using the convention of counting from the N-terminus of the mature protein) by the insertion of cysteine in place of the threonine of natural IL-3, but not at other positions.

Thus, this invention first provides cysteine added variants ("CAVs") of IL-3. CAVs of this invention encompass IL-3 muteins that contain at least one additional cysteine residue compared to the corresponding naturally occurring or previously known IL-3. The cysteine residue(s) are introduced into the peptide structure of the CAVs at one or more amino acid positions in the natural or previously known counterpart. In the case of human IL-3, we have determined that the naturally occurring cysteine residues at positions 16 and 84 form a disulfide bridge, essential to preserving the desired biological activity of the polypeptide. For the addition of novel cysteines, some positions within the polypeptide, such as position 15 and 51 are unsuitable; cysteines introduced at these positions give rise to human IL-3 polypeptides with substantially reduced biological activity. However, certain substitutions or deletions of residues 1-14 do not significantly diminish the desired biological activity of IL-3. Therefore, a preferred region of novel cysteine introduction into the polypeptide is within positions 1-14 inclusive. Currently, positions 6-12 inclusive are especially preferred sites for cysteine introduction. The subsequent attachment of sulfhydryl reactive compounds, including polymers, as discussed below, to the novel cysteines added at selected positions within this region will not result in any significant loss of biological activity.

By "cysteine added variant" as the term is used herein, we mean variants of IL-3 that are modified in amino acid structure relative to naturally occurring or previously known counterparts such that at least one cysteine residue is inserted into the natural or previously known sequence and/or is used to replace a different amino acid within that sequence.

Additionally, the natural IL-3 sequence, with an added initiator methionine for bacterial expression, may be further modified such that the first alanine is deleted at the N-terminus of the mature polypeptide, altering the amino terminal sequence from MET*ALA*PRO to MET*PRO (the "mp" mutein) For the "mp" mutein, such N-terminus modification permits more consistent removal of the N-terminal methionine. As is already known, in bacterial expression systems, cleavage at the N-terminal methionine occurs.

Alternatively, the natural IL-3 sequence may be further modified such that the first two amino acids at the N-terminus of the mature polypeptide are deleted, leaving a terminus beginning with MET*THR*GLN*THR* (the "m3" mutein). For the "m3" mutein, such N-terminus modification permits one to take advantage of the methionine at position 3 in the naturally occurring human IL-3 molecule, as the initiator methionine.

The CAVs of this invention make it possible to produce homogeneous, biologically active IL-3 compositions substantially specifically and consistently modified at selected positions with sulfhydryl reactive compounds (described hereinafter).

In the practice of this invention, at least one cysteine residue is introduced in that portion of the IL-3 polypeptide where modification via a sulfhydryl reactive compound is desired. The cysteine residue or residues are so introduced by genetic engineering methods as described below. Novel cysteine residues may be engineered into the polypeptide for example, by simple insertion of a cysteine codon into the DNA molecule at the desired site or by converting a desirably located asparagine or other codon to a cysteine codon. Convenient methods for site specific mutagenesis or DNA synthesis for producing a DNA molecule encoding the desired CAV, expression in procaryotic or eucaryotic host cells of the DNA molecule so produced, and recovery of the CAV produced by such expression are also disclosed.

The CAVs of this invention retain useful biological properties of the natural or previously known IL-3 and may thus be used for applications identified for the non-modified parent protein Modification with such sulfhydryl reactive compounds, however, is preferred Such biologically active, modified CAVs can be produced in homogeneous compositions which, it is contemplated, will provide improved pharmacokinetic profiles and/or solubility characteristics relative to the parent polypeptides. Furthermore, CAVs may enable the formation of multimeric forms of the normally monomeric polypeptide with the same, albeit improved characteristics. Multimeric CAVs also enable the formation of "hetero-conjugates"--i.e., two or more distinct polypeptides joined via the sulfhydryl groups of the added cysteine residue s, e.g ., IL-3 joined to EPO (erythropoietin) or IL-3 joined to G-CSF (granulocyte colony-stimulating factor).

Biological activity of the CAVs before or after modification with the sulfhydryl reactive compounds may be determined by standard in vitro or in vivo assays conventional for measuring activity of the parent polypeptide. Alternatively, we provide herein a "small scale" screening method wherein successful Cys modification and attachment of the sulfhydryl reactive compound may be tested.

Selective and homogeneous modification of the CAVs with sulfhydryl reactive compounds is possible since such compounds will covalently bond primarily only to the cysteine residue(s) in the CAV. Secondary reactivity at His, Lys and Tyr residue(s) may be observed, depending on the choice of sulfhydryl reactive compound, but at a significantly lower rate The modified CAVs so produced may then be recovered, and if desired, further purified and formulated into pharmaceutical compositions by conventional methods.

Sulfhydryl reactive compounds include compounds such as polyalkylene glycol, e.g. polyethylene and polypropylene glycol, as well as derivatives thereof, with or without coupling agents or derivatization with coupling or activating moieties, for example, with thiol, triflate, tresylate, aziridine or oxirane, or preferably with S-pyridyl or maleimide moieties. Compounds such as S-Pyridyl Monomethoxy PEG and Maleimido Monomethoxy PEG are exemplary. Additionally, sulfhydryl reactive compounds include, but are not limited to, charged or neutral polymers of the following types dextran, colominic acids or other carbohydrate based polymers, polymers of amino acids and biotin derivatives, resulting in a protein modified with this well known affinity reagent often used for antibody based assays.

Briefly, the method comprises reacting the CAV with a sulfhydryl reactive compound under suitable conditions, preferably non-denaturing conditions, and in sufficient amounts permitting the covalent attachment of the sulfhydryl reactive compound to the introduced cysteine residue(s) present in the polypeptide backbone of the CAV. The reaction may be reducible or non-reducible; and generally, the amount of sulfhydryl reactive compound used should be at least equimolar to the number of cysteines to be derivatized, although use of excess sulfhydryl reactive compound is preferred, both to improve the rate of reaction and to insure consistent modification at all reactive sites. The modified CAV produced, may then be recovered, purified and formulated by conventional methods. See e.g., WO 87/00056 and references cited therein

Other aspects of the present invention include therapeutic methods of treatment and therapeutic compositions which employ the modified IL-3 CAVs of the present invention, either alone or with other lymphokines, hematopoietins and/or growth factors, such as granulocyte macrophage colony-stimulating factor (GM-CSF), G-CSF, macrophage colony-stimulating factor (M-CSF), EPO, IL-1, IL-2, IL-4, IL-5, and IL-6. These methods and compositions take advantage of the improved pharmacokinetic properties of these modified CAVs to provide treatments, e.g., such as employing lower dosages of polypeptide, less frequent administration, lower immunogenicity and more desirable distribution, required for the therapeutic indications for the natural polypeptide.

Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of the invention, including illustrative examples of the practice thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the human IL-3 gene construct for E. coli expression, having the polypeptide sequence shown of natural (wild type) human IL-3, plus an initiator methionine, as expressed in E. coli, with the amino acids numbered from the N-terminus for reference to the muteins discussed herein

FIG. 2 is the "mp" modified polypeptide sequence of the IL-3 of FIG. 1, having the N-terminus modified in accordance with this invention as shown, with amino acids numbered for reference

FIG. 3 is the "m3" modified polypeptide sequence of the IL-3 of FIG. 1, having the N-terminus modified in accordance with this invention as shown with amino acids numbered for reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves the selective modification of IL-3 for pharmaceutical use, to both enhance its pharmacokinetic properties and provide homogeneous compositions for human therapeutic use. Although human IL-3, DNA and peptide sequences are preferred as the starting point in this invention, any primate IL-3 is susceptible to use in the method of the invention, given the significant homology between e.g., human and gibbon species of the protein and DNA. See Leary et al., Blood (1982) 70: 1343-1348. The method for selectively modifying primate IL-3 involves selecting locations in the polypeptide sequence for the attachment of sulfhydryl reactive compounds. This step may be accomplished by altering the amino acid sequence of the polypeptide by inserting cysteine residues at selected sites or by converting selected endogenous residues into cysteine residues. For example, the codons AAA or AAG, which code for lysine, can be changed to the codon TGC or TGT, which code for cysteine.

CAVs in accordance with this invention also include allelic variations in the IL-3 sequence, i.e. sequence variations due to natural variability from individual to individual, or with other amino acid substitutions or deletions which still retain desirable biological properties of the parent.

All CAVs of this invention may be prepared by expressing recombinant DNA sequences encoding the desired variant in host cells, e.g. procaryotic host cells such as E. coli, or eucaryotic host cells such as yeast or mammalian host cells, using methods and materials, e.g. vectors, as are known in the art. Host cells containing and capable of expressing the CAV-encoding DNA are thus emconpassed by this invention. DNA sequences encoding the variants may be produced synthetically or by conventional site-directed mutagenesis of DNA sequences encoding the protein or polypeptide or analogs thereof. FIG. 1 shows the human IL-3 gene construct inserted in plasmid pAL-hIL3-781 and expressed in the E. coli K12 strain designated GI586. This strain containing the plasmid was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA on Apr. 19, 1989 and given accession number 67932. Other DNA sequences for natural primate IL-3 have been cloned and the DNA sequences, including cDNA sequences, and specific peptide sequences for the same have been published, in PCT application number U.S. 87/01702, published as WO 88/00598 on Jan. 28, 1988, and are therefore known in the art. These DNA sequences have been deposited with the American Type Culture Collection and given accession numbers ATCC 67154, 67326, 67319 and 40246.

DNA molecules encoding natural human IL-3s therefore may be obtained (i) by cloning in accordance with the published methods, (ii) from the deposited plasmids, or (iii) by synthesis, e.g. using overlapping synthetic oligonucleotides based on the published sequences which together span the desired coding region. Such methods are known in the art. See the foregoing PCT application published as WO 88/00598 and PCT application number U.S.88/00402 published as W088/06161.

As mentioned above, DNA sequences encoding individual CAVs of this invention may be produced synthetically or by conventional site-directed mutagenesis of a DNA sequence encoding the parental IL-3 or analogs thereof. Such methods of mutagenesis include the M13 system of Zoller and Smith, Nucleic Acids Res. (1982) 10:6487-6500; Methods Enzymol. (1983) 100:468-500; and DNA (1984) 3:479-488, which uses single stranded DNA and the method of Morinaga et al., Bio/technology (July 1984) 636-639, which uses heteroduplexed DNA. Exemplary oligonucleotides used in accordance with such methods are described below. It should be understood, of course, that DNA encoding each of the CAVs of this invention may be analogously produced by one skilled in the art through site-directed mutagenesis using appropriately chosen oligonucleotides.

The new DNA sequences encoding the CAVs of this invention can be introduced into appropriate vectors for heterologous expression in the desired host cells, whether procaryotic or eucaryotic. The activity produced by the transiently transfected or stably transformed host cells (or their progeny) may be measured by using standard assays conventional for the parental protein. Where the host cell is bacterial, the DNA should be free of introns, e.g. a cDNA or synthetic DNA, and may be free of any secretory leader sequence. For eucaryotic expression, introns may be present or absent and a secretory leader sequence should preferably be present.

The CAVs produced by expression in the genetically engineered host cells may then be purified, and if desired formulated into pharmaceutical compositions by conventional methods, often preferably by methods which are typically used in purifying and/or formulating the parental protein. It is contemplated that such pharmaceutical compositions containing the CAV in admixture with a pharmaceutically acceptable carrier will possess similar utilities to those of the parental proteins, such as those set forth in WO 88/00598 supra, at page 3.

In another, and preferred, aspect of this invention, the CAVs produced by recombinant means as mentioned above are reacted with the desired sulfhydryl reactive compound under conditions permitting attachment of the sulfhydryl reactive moiety to the sulfhydryl group of the introduced cysteine residues in the peptide backbone of the CAV. These modified CAVs, preferably produced initially on a small scale, may then be screened for bioactive muteins possessing the sulfhydryl reactive compounds attached to the site or sites desired. Alternatively, this screening may be accomplished before attachment with the sulfhydryl reactive compound.

The term "sulfhydryl reactive compound" is defined herein as any compound having, or capable of being activated to have, a reactive group capable of forming a covalent attachment to the sulfhydryl group (-SH) of the cysteine residue. Included among such compounds are polymers such as PEG and polypropylene glycol (PPG), dextran, colominic acids or other carbohydrate based polymers and polymers of amino acids and biotin derivatives. Activation may occur by modification of the compound with a sulfhydryl moiety, such as a sulfhydryl group, thiol, triflate, tresylate, aziridine or oxirane, or preferably, with S-pyridyl or maleimide. The sulfhydryl reactive compound need not have any particular molecular weight, but a molecular weight of between about 1,000 and 30,000 for the activated compound is preferred, especially for PEG. Methods of attachment will be described in detail below By controlling the number and location of the cysteines in the CAV sequence, the number and location(s) of the attached sulfhydryl reactive compound can be selectively controlled Such control of attachment location and number enables the production of only certain selectively modified molecules retaining the desired biological activity, rather than production of a heterogeneous mixture of variably modified molecules, only some of which may be active. It is also important to note that this positional selectivity of the PEGylation or other attachment allows the normal functional interactions of the protein to be preserved, blocked, or regenerated by release of the sulfhydryl reactive compound.

Another aspect of the invention is therefore homogeneous compositions of modified IL-3 CAVs as described herein, e.g. PEGylated CAVs. Specific embodiments of IL-3 CAVs of the invention include human IL-3 which has a cysteine residue replacing lysine at position 10 and the m3 initiation sequence. (Amino acid numbers for the CAVs of the present invention are used herein in the conventional manner, sequentially from the N-terminus, and correlate with the numbering system used in FIG. 1 for the natural human IL-3 as expressed in E. coli.) Similarly, the naturally occurring lysine residue in human IL-3 (FIG. 1) at amino acid position 100 may be converted to a cysteine to create a human IL-3 CAV of the invention. Another embodiment has cysteine at positions 9 and 10 and the m3 initiation sequence.

For bacterial expression where the secretory leader-encoding DNA sequence is removed from the CAV-encoding DNA, it may be desirable to additionally modify the sequence such that it encodes an N-terminus comprising Met-Pro- - - (the mp mutein) instead of other N-termini such as Met-Ala-Pro. Such N-terminal modification permits more consistent removal of the N-terminal methionine. Alternatively, the first two residues of natural, human IL-3 may be deleted, leaving the naturally occurring methionine at position 3 as the translation initiator (the m3 mutein).

CAVs of this invention, modified as described, encompass CAVs containing other modifications as well, including truncation of the peptide sequence, deletion or replacement of additional amino acids with amino acids other than cysteine, insertion of new N-linked glycosylation sites, abolishment of natural N-linked glycosylation sites, etc., so long as the bioactivity of the molecule is retained. Thus, this invention encompasses CAVs encoded for by DNA molecules which are capable of hybridizing under stringent conditions to the DNA molecule encoding the parental IL-3 (or would be so capable but for the use of synonymous codons) so long as the encoded polypeptide contains one or more additional introduced cysteine residues relative to the parental peptide sequence.

Because the method and compositions of this invention provide homogeneous modified IL-3s, the invention also encompasses such homogeneous compositions for pharmaceutical use which comprise a therapeutically effective amount of a modified CAV described above in admixture with a pharmaceutically acceptable carrier. Such composition can be used in generally the same manner as that described for the natural or recombinant polypeptides. It is contemplated that the compositions will be used for treating a variety of conditions, e.g. involving stimulating hematopoietis or improving a patient's hematological profile. For example, a modified IL-3 of the present invention may be used as an adjunct to cancer chemotherapy or in the treatment of immune disorders, as discussed in WO 88/00598, at page 17-19. The exact dosage and method of administration will be determined by the attending physician depending on the particular modified CAV employed, the potency and pharmacokinetic profile of the particular compound as well as on various factors which modify the actions of drugs, for example, body weight, sex, diet, time of administration, drug combination, reaction sensitivities and severity of the particular case. Generally, the daily regimen should be in the range of the dosage for the natural or recombinant unmodified IL-3, e.g. a range of about 0.1 to about 100 μg of polypeptide per kilogram of body weight, preferably from about 0.1 to about 30 μg of polypeptide per kilogram of body weight.

The therapeutic method and compositions of the present invention may also include co-administration with other drugs or human factors. A non-exclusive list of other appropriate hematopoietins, CSFs (colony stimulating factors) and interleukins for simultaneous or serial co-administration with the CAVs of the present invention includes GM-CSF, CSF-1 (in its various known forms; CSF-1 is also referred to as M-CSF or macrophage colony-stimulating factor), G-CSF, Meg-CSF, EPO, IL-1, IL-2, IL-4, IL-6, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor. Additionally, the CAVs of the present invention may be administered with, or chemically attached to, monoclonal or polyclonal antibodies in a therapeutic use. Alternatively, these growth factors may be attached to certain toxins, e.g., ricin, for use in a therapeutic regimen. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition or regimen. In the case of pharmaceutical compositions containing modified lymphokine CAVs, for example, progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g. white cell count, hematocrit and the like.

The following examples illustratively describe the CAVs and the methods and compositions of the present invention.

EXPERIMENTAL MATERIALS, METHODS AND EXAMPLES EXAMPLE 1 Eucaryotic Expression Materials and Methods

Eukaryotic cell expression vectors into which DNA sequences encoding CAVs of this invention may be inserted (with or without synthetic linkers, as required or desired) may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufman et al., J. Mol. Biol., (1982) 159:601-621; Kaufman, Proc. Natl. Acad. Sci. (1985) 82:689-693. See also WO 87/04187, filed Jan. 2, 1987 (pMT2 and pMT2-ADA), and U.S. patent application Ser. No. 88,188, filed Aug. 21, 1987 now abandoned (pxMT2). Exemplary vectors useful for mammalian expression are also disclosed in the patent applications cited in Example 4, which are hereby incorporated by reference. Eucaryotic expression vectors useful in producing variants of this invention may also contain inducible promoters or comprise inducible expression systems as are known in the art. See U.S. patent application Ser. No. 893,115 (filed Aug. 1, 1986) now abandoned and PCT/U.S.87/01871, published as WO88/00975 on Feb. 11, 1988.

Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as hematopoietic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.

If eucaryotic host cells are used, they will preferably will established mammalian cell lines. For stable integration of the vector DNA into chromosomal DNA, and for subsequent amplification of the integrated vector DNA, both by conventional methods, CHO (Chinese Hamster Ovary) cells are presently preferred in such embodiments Alternatively, the vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al., Cell (1984) 36: 391-401) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other usable mammalian cell lines include HeLa, COS-1 monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines and the like.

Stable transformants then are screened for expression of the CAV product by standard immunological or activity assays. The presence of the DNA encoding the CAV IL-3s may be detected by standard procedures such as Southern blotting. Transient expression of the CAV genes during the several days after introduction of the expression vector DNA into suitable host cells such as COS-1 monkey cells is measured without selection by activity or immunologic assay of the proteins in the culture medium.

Following the expression of the DNA by conventional means, the CAVs so produced may be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods.

EXAMPLE 2 Bacterial and Yeast expression

Bacterial and yeast expression may be effected by inserting (with or without synthetic linkers, as required or desired) the DNA molecule encoding the desired CAV into a suitable vector (or inserting the parental DNA sequence into the vector and mutagenizing the sequence as desired therein), then transforming the host cells with the vector so produced using conventional vectors and methods as are known in the art, e.g. as disclosed in published PCT Application No. WO 86/00639, published Jan. 30, 1986. Transformants are identified by conventional methods and may be subcloned if desired. Characterization of transformants and recombinant product so produced may be effected and the product recovered and purified, all as described in Example 1.

For bacterial expression, the DNA sequences encoding the CAVs are preferably modified by conventional procedures to encode only the mature polypeptide and may optionally be modified to include preferred bacterial codons.

Expression in E. coli

The CAVs of Example 5 were expressed in E. coli as follows: Plasmid pAL-hIL3-781 was transformed into an E. coli K12 strain GI586, a derivative of strain W3110 in which the CI and Rex regions of bacteriophage lambda carrying the CI 857 allele have been inserted into the ClaI site of the lacZ gene of the bacterial genome. This insert consists of all of the DNA sequences between nucleotides 35711 and 38104 of the phage genome. See F. Sanger, et al., J. Mol. Biol. (1982) 162:729. E. coli K12 strain GI586 (pAL-hIL3-781) was deposited at the ATCC on Apr. 19, 1989 and given accession number 67932.

When GI586 transformed with pAL-hIL3-781 is grown at 30 degrees centigrade to high cell density and then heated to 40 degrees centigrade, IL-3 is produced rapidly and accumulates over the next two or three hours to reach greater than 10 percent of the total cellular protein. This protein is produced in an insoluble form which must be solubilized and refolded by conventional methods See, e.g., T. E. Creighton, Prog. Biophys. Molec. Biol. (1978) 33:231-297. Following expression, the CAVs so produced were recovered, purified and characterized as follows.

1. Purification of CAV IL-3

All buffers were prepared using glass distilled water; all were degassed for at least five minutes, using house vacuum/sonification, prior to the addition of DTT.

First, 400 grams wet weight frozen E. coli cell paste was suspended in 2500 ml of buffer containing 50 mM Tris-HCl, pH 8.5, 1 mM EDTA, 5 mM P-aminobenzamidine, 1 mM PMSF and 2 mM DTT (hereinafter in this Example "buffer A"), to obtain a final volume of 2850 ml. Glass rods and magnetic stirrers were used to resuspend the cell paste. Then the cell suspension was lysed by passing it through a matin gaulin valve at 9000 psi four times, with cooling between each time. Temperature was maintained below 30 degrees centigrade by collection of the lysate into glass vessels cooled in ice/water mixture. Protein concentration was 22 mg/ml; final volume was 2850 ml.

The lysate was centrifuged for 30 minutes at 8000 rpm in a Sorval centrifuge with a GS-3 rotor. The supernatant (2600 ml at 17.0 mg/ml) was discarded and the resultant pellet (hereinafter in this Example P1) from this centrifugation was resuspended in approximately 400 ml buffer A, using glass rods and a magnetic stirrer. The milky suspension was then passed through an 18 gauge needle using a 60 ml syringe The final volume was 640 ml, with a protein concentration of 25.6 mg/ml.

The resuspended P1 pellet was then centrifuged for 10 minutes in a Sorval centrifuge with a GS-3 rotor at 8000 rpm. The supernatant from this centrifugation was poured into two fresh centrifuge tubes (hereinafter in this example "S2") and the resultant pellet ("P2") was resuspended in buffer A to a final volume of 165 ml, with a protein concentration of 50 mg/ml. The S2 supernatant was then centrifuged for 10 minutes and the resulting pellet ("P3") was resuspended in 65 ml buffer A. The resulting supernatant ("S3") was further centrifuged for 10 minutes and the resulting P4 pellet was resuspended in buffer A to a final volume of 50 ml, with a protein concentration of 11.3 mg/ml. Because the P4 pellet contained so little IL-3, it was not used in subsequent steps. The S4 supernatant from the final centrifugation, approximately 600 ml, contained the membranous components at a concentration of approximately 10 mg/ml.

The P2 and P3 pellets were pooled and centrifuged at 9000 rpm (GSA rotor) for 10 minutes yielding two pellets ("P2-2") and a cloudy supernatant, which using HPLC analysis was found void of IL-3 and was discarded. The P2-2 pellet was frozen at -20 degrees centigrade for later use.

The frozen P2-2 pellet was then resuspended in buffer A (which contained 10 mM DTT rather than 2 mM DTT) to a final volume of 100 ml using glass rods and magnetic stirrer and then passed through an 18 gauge needle. 400 ml of 7M fresh guanidine in the 10 mM DTT buffer A was added to the resuspended P2-2 and after one quick inversion, the solubilized P2-2 pellet was immediately placed in 3×250 centrifuge tubes and centrifuged for 15 minutes at 8000 rpm (GSA rotor). 500 ml of the supernatant at a concentration of 5.98 mg/ml was purified further at room temperature by RP-HPLC. The foregoing two steps were performed in 17 to 22 minutes

2. RP-HPLC separation of IL-3 CAVs

The buffers used in this separation protocol were 0.1% (v/v) TFA in water, and 0.1% TFA in acetonitrile.

A two inch Vydac C4 column was equilibrated in 10% acetonitrile. The supernatant from the 7M guanidine solubilization was immediately applied onto the C4 column having a volume of approximately 470 ml at 180 ml per minute. The column was developed at 20 ml per minute and was washed in 10% acetonitrile until absorbance at 280 nm was back to baseline. The following gradient was established by washing with the following concentrations of acetonitrile at the following times:

______________________________________time (in minutes)           % acetonitrile______________________________________5               1010              3555              5560              8065              8067.5            10______________________________________

40 ml fractions were collected after 35 minutes into the gradient. 10 μl samples were removed from each fraction, vacuum speed dried and taken up in 20 μl of 2x SDS-sample Laemmli buffer. SDS-PAGE analysis was performed and IL-3 presence was confirmed. All fractions were then frozen at -80 degrees centigrade.

3. Refolding of IL-3 CAVs

One of the RP-HPLC separated fractions containing approximately 75 mg (7.5 ml) IL-3 was diluted to approximately 0.5 mg/ml by the addition of 142.5 ml of 6.4M guanidine in 50 mM NaPO4 7.0, 1mM EDTA and 0.2 mM DTT. The mixture was then added to 750 ml of 50 mM Na PO4 pH7, 1 mM EDTA, 0.2 mM DTT buffer, transferred to dialysis tubing and dialyzed for two hours against 4 L of the same buffer. The IL-3 (now approximately 0.22M guanidine) was twice further dialyzed against 8 L of the same buffer containing 0.1 mM DTT.

PEGylation of this purified IL-3 is set forth in Examples 7 and 8 below.

4. Confirmation of bioactivity

Bioactivity of the IL-3 CAV may be confirmed by using the TF-1 cell proliferation assay. The TF-1 cell line has been described (Kitamura et al., Blood (1989) 73:375-380). Cells are maintained at 37 degrees centigrade in humid air containing 5% CO2 and culture media used is RPMI (Gibco), 10% heat inactivated fetal calf serum, 2 mM L-glutamine, with 5 ng/ml of recombinant GM-CSF added. Every 3-4 days cells are adjusted to a density of 2×105 cells/ml Just prior to assay the cells are centrifuged 500xG, 5 minutes, washed in culture media without rGM-CSF, recentrifuged and resuspended at a density of 105 cells/ml.

IL-3 samples to be assayed are diluted between 1:500 and 1:10,000 in culture media without rGM-CSF. 125 μl of the diluted sample is placed in the top row of a 96 well microtiter plate. The remaining wells are filled with 100 μl of culture media without rGM-CSF and the top row samples are serially diluted five fold down the microtiter plate. To each well, 100 μl of diluted cells (104 cells) are added and the plate is incubated at 37 degrees centigrade, 5% CO 2 for 48-72 hours. Thereafter, 0.5 uCi 3 H-thyidine is added per well and the plate is further incubated for 4-6 hours. Cells are then harvested using an automated cell harvester (LKB 1295-001) and the 3 H-thymidine uptake is quantitated.

Alternatively, a CML proliferation assay as described in PCT/U.S.87/017024, International Publication Number WO88/00598, published Jan. 28, 1988, can be used.

EXAMPLE 4 Mutagenesis Protocol

Site directed mutagenesis may be effected using conventional procedures known in the art. See e.g., International Applications Nos. WO 87/07144, and WO 87/04722, and U.S. patent application Ser. Nos. 099,938 (filed Sep. 23, 1987) now abandoned and 088,188 (filed Aug. 21, 1987) now abandoned and the references cited therein.

EXAMPLE 5 Exemplary human IL-3 Mutagenesis Reactions

The following human IL-3 muteins were engineered by substitution of the codons indicated for a cys codon, or by insertion of a cys codon, using conventional site directed mutagenesis techniques:

______________________________________mp mutein    m3 mutein   cys modification______________________________________mpCys10  m3cys10     AAA to TGC (Lys to Cys)mpCys6   m3Cys6      ACT to TGC (Thr to Cys)mpCys8   m3Cys8      TCT to TGC (Ser to Cys)mpCys12  m3Cys12     TCT to TGC (Ser to Cys)mpCys100 m3Cys100    AAG to TGT (Lys to Cys)mpCys134 m3Cys134    Insertion of TGT between                TTC and TAG (Cys between                Phe 133 and stop codon)mpCys3               ATG to TGC (Met to Cys)mpΔ1Cys19      Replacement of amino                acids 1-15 with the "mp"                terminus and modif. of                pos. 19 from ATG to TGC                (Met to Cys)    m3Cys6,10   ACT and AAA to TGC                (Thr and Lys to Cys)    m3Cys9,10   TTA and AAA to TGC                (Leu and Lys to Cys)    m3Cys6,8    ACT and TCT to TGC                (Thr and Ser to Cys)    m3Cys6,8,10 ACT, TCT and AAA to TGC                (Thr, Ser and Lys to Cys)    m3Cys8,9,10 TCT, TTA and AAA to TGC                (Ser, Leu and Lys to Cys)______________________________________

In the examples depicted above the modification site of the natural IL-3 protein is designated by the number after "Cys" and the amino acid sequence of the CAV is identical to that of the native protein, except for the position indicated, with respect to the N-terminus (see FIG. 1). The "mp" and "m3" designation signify the two different alterations of the N-terminus that will be discussed in detail below. Additionally, cys may be introduced in place of native codons at positions 63 or 66, alone or in combination with other cys introduction(s), e.g. at position 10--with any of the described N-termini.

With respect to IL-3 muteins, certain point modifications may result in partial loss of biological activity or inability of the sulfhydryl reactive compound to attach. For example, modification at position 28 results in a biologically active CAV, but attachment of a sulfhydryl reactive compound fails, possibly because position 28 appears internally in the refolded CAVs tertiary structure Compare, Wingfield, D., et al., Eur. J. Biochem. (1989) 179:565-571, in which the authors discussed the Cys modification of IL-1β at position 138 to active IL-1β-phycoerythrin conjugate Additionally, we have found that substitution of a cysteine residue for the amino acids at positions 15 or 51 of the natural human IL-3 may result in partial loss of bioactivity. To test for activity after attachment of the sulfhydryl reactive compound, this invention further provides a "small scale" screening technique to readily determine whether modification and attachment has been successful (see Example 9 below).

The human IL-3 was additionally modified at its N-terminus in two different and alternative configurations, represented by the "mp" and "m3" designations. The "mp" designation indicates a deletion of the first alanine in the natural human IL-3 protein, thereby changing the N-terminal sequence from MET*ALA*PRO to MET*PRO. Compare FIGS. 1 and 2. The "m3" designation indicates a deletion of the first two amino acids in the natural human IL-3 protein, MET*ALA*PRO, to yield a terminus beginning MET*THR*GLU*THR*. Compare FIGS. 1 and 3. The reasons for these modifications have already been discussed. With respect to N-terminus modification of the mpΔ1Cys19 mutein, amino acids 1-15 were deleted and replaced with the "mp" terminus.

It should be understood of course that the depicted list of muteins is merely exemplary and not exclusive. The design and synthesis of alternative and additional muteins of both human and gibbon IL-3 in accord with this invention is well within the present skill in the art. Synthesis of such muteins may be conveniently effected using conventional techniques and methods.

One skilled in the art, of course, could readily design and synthesize other muteins for substitution of cysteine codons or insertion thereof in DNA sequences encoding IL-3. To modify more than one site, mutagenesis may be carried out iteratively, or in some cases using an oligonucleotide designed for mutagenesis at more than one site.

EXAMPLE 6 Synthesis of DNA molecules encoding CAVs

As an alternative to the production of CAV-encoding DNA by mutagenesis of the parental IL-3 DNA sequence, it should be understood that the desired CAV-encoding DNA may be prepared synthetically. In that case, it will usually be desirable to synthesize the CAV IL-3 DNA in the form of overlapping oligonucleotides, e.g. overlapping 50-80 mers, which together span the desired coding sequence and contain the cysteine additions desired: ##STR1## Given a desired coding sequence, the design, synthesis, assembly and ligation, if desired, to synthetic linkers of appropriate oligonucleotides is well within the present level of skill in the art.

EXAMPLE 7 PEGylation of the IL-3 mpCys10 mutein

The mutein human IL-3 mpCys10 was prepared in accordance with Example 5 above and PEGylated with two PEG 5000 derivatives, S-Pyridyl Monomethoxy PEG 5000 and Maleimido Monomethoxy PEG 5000.

a. PEGylation with S-Pyridyl Monomethoxy PEG 5000: a reducible linkage

1. Preparation of the sulfhydryl reactive compound

PEG 5000 was activated for attachment to a sulfhydryl group as follows. 2.0 grams of Monomethoxy PEG 5000 amine was dissolved in 12 ml dry peroxide free, dioxane. 144 mg (15% excess) of N-succinimidyl-3-(2 pyridyldithio) propionate (SPDP) was added as a dry powder and the reaction was allowed to proceed at room temperature. After 24 hours, the S-pyridyl Monomethoxy PEG 5000 product was precipitated using dry, peroxide free diethyl ether and washed with ether. The product was dried under vacuum to obtain 1.92 grams of white solid, which was identified as S-Pyridyl Monomethoxy PEG 5000 by NMR and IR. The PEG 10,000 analog was likewise prepared via an analogous procedure.

2. PEGylation of mutein C10 human IL-3

For this coupling, natural (wild type) human IL-3 was also treated with the PEGylation reagents as a negative control. A stock solution at 1 mg/ml of the mpCys10 mutein in a pH 7 buffered solution of 50 mM NaH2 PO4, 100 micro M DTT, 1 mM EDTA and about 3mM Guanidine HCl was used. DTT was added to prevent dimerization of the protein; EDTA was added to prevent dimerization via metal mediated oxidative coupling. Guanidine remains as an artifact of the refolding of the protein. A pH 7 was used; a range of 6.5-7.5 is preferred. 0.9 mg of S-Pyridyl Monomethoxy PEG 5000, prepared as set forth above, was weighed into an eppendorf tube. 360 microliters of the buffered mutein was added and the mixture was vortexed briefly to homogeneity. The reaction was performed at 4 degrees centigrade and when sampled after 2 hours, was found to be complete. Analysis on a 10-20% gradient SDS acrylamide gel showed the product as nearly pure and running at about 28 kD. (By comparison, mpCys10 and its dimer were used as standards and found to migrate to 15 and 30 kD respectively.) A reducing lane on the gel showed that the PEGylated IL-3 mutein is sensitive to reduction by DTT and regenerated the original protein at about 15 kD.

b. PEGylation with Maleimido Monomethoxy PEG 5000: a non-reducible linkage.

1. Preparation of the sulfhydryl reactive compound

In this experiment, PEG 5000 activation was accomplished as follows. 2.0 mg of monomethoxy PEG 5000 amine was dissolved in 12 ml of dry, peroxide free dioxane. 154 mg of sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (15% excess) was added as a dry powder and the reaction was allowed to proceed at room temperature. After 24 hours, work up of the product was carried out in the same manner as the S-Pyridyl Monomethoxy PEG 5000 to obtain 1.82 grams Maleimido Monomethoxy PEG 5000. The PEG 10,000 analog was prepared similarly.

2. PEGylation of the cys10 IL-3 mutein

This reaction was carried out in the same manner as the PEGylation reaction using the reducible PEG 5000 reagent and with natural human IL-3 as a negative control. However, 1.0 mg of the PEG derived PEGylating agent Maleimido Monomethoxy PEG 5000 was used. 400 microliters of the mpCys10 IL-3 mutein was added and vortexed to homogeneity. At t=2 hours the reaction was found to be complete. The product was nearly pure and indistinguishable from the S Pyridyl derived conjugate in molecular weight. However, this product is perfectly inert to reductive conditions, such as DTT; in this reducing lane the product, at 28 kD, persists.

In both control reactions, nothing indicative of conjugation is evident at 2 hrs or even at 24 hrs. Selectivity for accessible sulfhydryls in this chemistry is therefore very high.

EXAMPLE 8 PEGylation of multiple cysteine muteins m3Cys9,10 and m3Cys6,10

In this experiment, protein stock for both muteins was at 300 μg/ml in the phosphate buffer solution, as described in Example 7. PEGylation stock solutions consisted of the S-pyridyl or Maleimide activated PEG 5000 polymers at 50 μg/ml in the same buffer. To initiate the reaction, 11 μl of the appropriate PEG stock was added to 100 μl of the appropriate protein stock (either the m3Cys9,10 mutein or the m3Cys6,10 mutein) while vortexing. Reactions were allowed to proceed at 4 degrees centigrade overnight. SDS gel analysis of the products as described above revealed that only a trace of starting material remained with both chemistries. Furthermore, both chemistries resulted in new products with a gel mobility of about 37 kD. Reducing lanes on this same gel show that the maleimide conjugate is resistant to reducing, while the S-Pyridyl derived conjugate reverts to starting material.

EXAMPLE 9 Screening of novel CAVs

Having the constructed novel DNA molecules encoding CAVs in the appropriate expression vector and having attached the sulfhydryl reactive compound to the muteins, it may be desirable to produce each CAV protein on a small scale and "screen" for muteins which possess the desired attachment site or sites. The biological activity of each CAV, before and after attachment of the sulfhydryl reactive compound can be rapidly assessed using an in vitro assay.

Small scale bacterial production of CAV muteins

Bacterial strain GI586 was transformed with purified plasmid DNAs consisting of bacterial expression vector, pAL-hIL3-781, ATCC Accession Number 67932, with novel CAV IL-3 coding sequences The transformed cells were spread on LB agar plates containing 50 μg/ml ampicillin at a density to yield approximately 100 colonies per plate. 3 ml of L broth plus 50 μg/ml ampicillin was inoculated with a single bacterial colony and grown overnight at 30 degrees centigrade. 50 ml of induction media (0.1×L Broth, 1×M9 salts, 0.4% glucose, 1 mM MgSO4, 50 μg/ml ampicillin) was inoculated with 1 ml of the overnight culture. The 50 ml culture was grown with aeration at 30 degrees centigrade until an 0.5 OD 600 nm level was reached, then the temperature was shifted to 40 degrees centigrade and growth continued for at least 2 hours.

Cells were then harvested by centrifugation at 3500 rpm for minutes in a Sorval centrifuge with a 3B rotor. The supernatant was discarded and the cell pellet resuspended in 1 ml of buffer PED (50 mM NaH2 PO4, pH 7.0, 1 mM EDTA, 5mM DTT, 10 mM PMSF). This 1 ml solution was passed twice through a French Press at 10,000 psi and kept on ice. The solution was then microfuged for 5 minutes at 12,000 rpm. The supernatant was discarded and the pelleted material was resuspended in 150 μl of 7M guanidine-HCl in PED buffer The solution was then diluted with 650 μl of PED buffer and placed in dialysis tubing (10,000 MWCO Spectrapore) The sample was dialyzed for at least 4 hours against 2 liters of PED.1 buffer (50 mM NaH2 PO4 pH 7.0, 1 mM EDTA, 0.1 mM DTT). The sample was collected and microfuged to remove precipitated proteins. The sample was then analyzed on a 12% Laemmli SDS PAGE gel and the amount of IL-3 protein estimated.

The protein solution was then concentrated to about 0.5 mg/ml, and 200 μg was reacted with a 15 fold molar excess of either S-Pyridyl Monomethoxy PEG 5000 or Maleimido Monomethoxy PEG 5000 for several hours at 4 degrees centigrade. The products were then analyzed by SDS PAGE and biological activity determined by an in vitro TF-1 cell proliferation assay.

Alternatively, this small scale production for screening may be carried out before attachment of the sulfhydryl reactive compound. In that case, biological activity may still be determined by an in vitro TF-1 cell proliferation assay and the products may be analyzed by SDS PAGE analysis, in accordance with known techniques.

The same or similar procedures may be used by one skilled in the art to attach other sulfhydryl reactive compounds to the other CAVs of the invention Homogeneity can be observed by conventional analysis of the modified CAVs so produced e.g. using standard SDS-PAGE or HPLC analysis.

Numerous modifications may be made by one skilled in the art to the methods and compositions of the present invention in view of the disclosure herein Such modifications are believed to be encompassed by this invention as defined by the appended claims

Claims (9)

We claim:
1. A cysteine added variant of human interleukin-3 characterized by having an added cysteine residue in the N-terminal region between amino acids 1 through 14 of the natural human interleukin-3 sequence, said added cysteine residue either being substituted for an amino acid in the natural human interleukin-3 sequence or being inserted between two amino acids of the natural human interleukin-3 sequence, said cysteine added variant being further characterized by having a polyethylene glycol moiety covalently attached to said added cysteine residue.
2. A cysteine added variant of claim 1 wherein said added cysteine residue is located in the N-terminal region between amino acids 6 through 12 of the natural human interleukin-3 sequence.
3. A cysteine added variant of claim 2, further characterized by having an N-terminal commencing with methionine and the deletion of alanine at position 1 of the natural human interleukin-3.
4. A cysteine added variant of claim 3 wherein said cysteine residue is substituted for the serine residue at position 8 of the natural human interleukin-3 sequence.
5. A cysteine added variant of claim 3 wherein said cysteine residue is substituted for the lysine at position 10 of the natural human interleukin-3 sequence.
6. A cysteine added variant of claim 2, further characterized by having an N-terminus modified by the deletion of alanine and proline at positions 1 and 2 of the natural human interleukin-3.
7. A pharmaceutical composition for stimulating hematopoiesis comprising a therapeutically effective amount of a cysteine added variant of claim 1 or claim 4 in admixture with a pharmaceutically acceptable carrier.
8. The composition of claim 7 wherein said polyethylene glycol moiety has a molecular weight of about 1,000 to about 30,000.
9. A method of producing a cysteine added variant of claim 1 comprising covalently attaching said polyethylene glycol moiety to said added cysteine residue of said cysteine added variant, produced by culturing a host cell containing and capable of expressing a DNA sequence encoding said cysteine added variant.
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EP19900907849 EP0469074B1 (en) 1989-04-21 1990-04-19 Cysteine added variants il-3 and chemical modifications thereof
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Cited By (136)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013316A1 (en) * 1992-12-16 1994-06-23 Board Of Regents, The University Of Texas System Potent and specific chemically-conjugated immunotoxins
US5359035A (en) * 1985-12-21 1994-10-25 Hoechst Aktiengesellschaft Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF)
WO1995011987A1 (en) * 1993-10-29 1995-05-04 Incyte Pharmaceuticals, Inc. Chimeric proteins including protease nexin-1 variants
US5536495A (en) * 1994-04-15 1996-07-16 Foster; Preston F. Use of G-CSF to reduce acute rejection
US5604116A (en) * 1992-11-24 1997-02-18 G. D. Searle & Co. Interleukin-3 (IL-3) multiple mutation polypeptides, recombinant production of the same, and corresponding therapeutic methods
EP0785276A1 (en) * 1994-09-29 1997-07-23 Ajinomoto Co., Inc. Modification of peptide and protein
US5705362A (en) * 1992-05-25 1998-01-06 Gist-Brocades, N.V. Modified signal sequences
US5718893A (en) * 1984-04-15 1998-02-17 Foster; Preston F. Use of G-CSF to reduce acute rejection
US5747027A (en) * 1995-04-07 1998-05-05 The Regents Of The University Of California BH55 hyaluronidase
US5763196A (en) * 1996-01-26 1998-06-09 Boehringer Mannheim Corporation Assays using cross-linked polypeptide fragments of β-galactosidase
US5766897A (en) * 1990-06-21 1998-06-16 Incyte Pharmaceuticals, Inc. Cysteine-pegylated proteins
US5824778A (en) * 1988-12-22 1998-10-20 Kirin-Amgen, Inc. Chemically-modified G-CSF
US5830761A (en) * 1995-06-07 1998-11-03 Genetics Institute, Inc. Medium and methods for culturing mammalian cho cells
US5846770A (en) * 1994-11-22 1998-12-08 Genetics Institute, Inc. DNA molecules encoding human chordin
US5856138A (en) * 1991-08-07 1999-01-05 Takeda Chemical Industries, Ltd. Human parathyroid hormone muteins and production thereof
WO1999003887A1 (en) * 1997-07-14 1999-01-28 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US5880255A (en) * 1988-10-20 1999-03-09 Polymasc Pharmaceuticals Plc Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct of PEG and granulocyte-macrophage colony stimulating factor
US5883230A (en) * 1990-08-29 1999-03-16 Genetics Institute, Inc. Multidomain hematopoiesis stimulators
US5932427A (en) * 1993-11-10 1999-08-03 Henry M. Jackson Foundation For The Advancement Of Military Medicine In vitro assay system for identifying compositions useful for stimulating B cells
US5935852A (en) * 1997-07-03 1999-08-10 Genetics Institute, Inc. DNA molecules encoding mammalian cerberus-like proteins
US5945397A (en) * 1989-09-05 1999-08-31 Immunex Corporation Purified p75 (type II) tumor necrosis factor receptor polypeptides
US5951972A (en) * 1990-05-04 1999-09-14 American Cyanamid Company Stabilization of somatotropins and other proteins by modification of cysteine residues
US5952226A (en) * 1996-11-05 1999-09-14 Modex Therapeutiques Hypoxia responsive EPO producing cells
US5976783A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Bis-maleimido cross-linking agents
US5976857A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Cross-linked polypeptide fragments of β-galactosidase
WO1999055377A2 (en) * 1998-04-28 1999-11-04 Applied Research Systems Ars Holding N.V. Polyol-ifn-beta conjugates
US6017523A (en) * 1995-06-06 2000-01-25 G.D. Searle & Co. Therapeutic methods employing mutant human interleukin-3 (IL-3) polypeptides
US6074639A (en) * 1992-11-24 2000-06-13 G. D. Searle & Co. Ex vivo expansion of hematopoietic cells using interleukin-3 (IL-3) variant fusion proteins
WO2000042175A1 (en) 1999-01-14 2000-07-20 Bolder Biotechnology Inc. Methods for making proteins containing free cysteine residues
US6143866A (en) * 1989-07-18 2000-11-07 Amgen, Inc. Tumor necrosis factor (TNF) inhibitor and method for obtaining the same
US6306820B1 (en) 1996-12-06 2001-10-23 Amgen Inc. Combination therapy using a TNF binding protein for treating TNF-mediated diseases
US6323006B1 (en) * 1996-12-18 2001-11-27 Universidade Federal De Minas Gerais Recombinant human beta-CIS interferon
US6384195B1 (en) * 1988-10-20 2002-05-07 Polymasc Pharmaceuticals Plc. Process for fractionating polyethylene glycol (PEG) —protein adducts and an adduct of PEG and granulocyt-macrophage colony stimulating factor
US6420339B1 (en) * 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
US6458355B1 (en) 1998-01-22 2002-10-01 Genentech, Inc. Methods of treating inflammatory disease with anti-IL-8 antibody fragment-polymer conjugates
WO2002077218A1 (en) 2001-03-22 2002-10-03 Novo Nordisk Health Care Ag Coagulation factor vii derivatives
US6468532B1 (en) 1998-01-22 2002-10-22 Genentech, Inc. Methods of treating inflammatory diseases with anti-IL-8 antibody fragment-polymer conjugates
US20020177688A1 (en) * 1988-12-22 2002-11-28 Kirin-Amgen, Inc., Chemically-modified G-CSF
US20030017883A1 (en) * 2001-05-28 2003-01-23 Hiroaki Yoshiike Swing analyzing device
US20030021790A1 (en) * 1998-01-22 2003-01-30 Genentech, Inc. Antibody fragment-polymer conjugates and humanized anti-IL-8 monoclonal antibodies
US20030044908A1 (en) * 2001-03-22 2003-03-06 Egon Persson Coagulation factor VII derivatives
US20030053982A1 (en) * 1994-09-26 2003-03-20 Kinstler Olaf B. N-terminally chemically modified protein compositions and methods
US6541620B1 (en) 1989-07-18 2003-04-01 Angen Inc. Nucleic acids encoding TNF inhibitor and method of production
US6552170B1 (en) * 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
WO2003040695A2 (en) * 2001-11-08 2003-05-15 Moyle William R Protein knobs
US20030130193A1 (en) * 1999-01-29 2003-07-10 Bailon Pascal Sebastian GCSF conjugates
US6600017B1 (en) * 1997-08-14 2003-07-29 The Regents Of The University Of California Fluorescent amyloid Aβ peptides and uses thereof
US6602498B2 (en) 2000-02-22 2003-08-05 Shearwater Corporation N-maleimidyl polymer derivatives
US20030171284A1 (en) * 1997-07-14 2003-09-11 Cox George N. Derivatives of growth hormone and related proteins, and methods of use thereof
US20040087778A1 (en) * 1998-10-23 2004-05-06 Amgen Inc. Modified peptides as therapeutic agents
US20040126361A1 (en) * 2002-12-26 2004-07-01 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency
US20040136952A1 (en) * 2002-12-26 2004-07-15 Mountain View Pharmaceuticals, Inc. Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
US20040181035A1 (en) * 1994-10-12 2004-09-16 Amgen, Inc. N-terminally chemically modified protein compositions and methods
US20040186051A1 (en) * 2001-10-02 2004-09-23 Kelley Robert F Apo-2 ligand variants and uses thereof
US20040204548A1 (en) * 2002-12-31 2004-10-14 Antoni Kozlowski Hydrolytically stable maleimide-terminated polymers
US20040225097A1 (en) * 2003-05-07 2004-11-11 Kwang Nho Novel preparation method of peg-maleimide derivatives
US6818435B2 (en) 2000-05-15 2004-11-16 Tecan Trading Ag Microfluidics devices and methods for performing cell based assays
US20050026834A1 (en) * 1999-01-14 2005-02-03 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US20050058621A1 (en) * 1997-07-14 2005-03-17 Cox George N. Derivatives of growth hormone and related proteins, and methods of use thereof
US6870033B1 (en) 1997-02-21 2005-03-22 Genentech, Inc. Antibody fragment-polymer conjugates and humanized anti-IL-8 monoclonal antibodies
US20050090520A1 (en) * 2003-09-12 2005-04-28 Per Lindquist Treatment of disease or injury of the nervous system with FTY720
US20050143563A1 (en) * 2002-03-20 2005-06-30 Myung-Ok Park Preparation of g-csf stoichiometrically conjugated with biocompatible polymers at cystein residue
US20050163782A1 (en) * 2003-06-27 2005-07-28 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US20050176922A1 (en) * 2003-12-03 2005-08-11 Mcmanus Samuel P. Methods of preparing maleimide functionalized polymers
US20050250696A1 (en) * 1996-07-09 2005-11-10 Fisher Eric F Truncated soluble tumor necrosis factor type-I and type-II receptors
US6984623B2 (en) 1993-12-07 2006-01-10 Genetics, Institute Institute, LLC. Tendon-inducing compositions
US20060009381A1 (en) * 2004-07-07 2006-01-12 I.P. Randywyck B.V. Annexins, derivatives thereof, and annexin-cys variants, as well as therapeutic and diagnostic uses thereof
US20060029573A1 (en) * 2004-06-30 2006-02-09 Chun Shen Pegylated interferon alpha-1b
US20060083683A1 (en) * 1999-01-21 2006-04-20 Vanessa Hsei Antibody fragment-polymer conjugates and uses of same
US20060177894A1 (en) * 2002-11-08 2006-08-10 Moyle William R Protein knobs
US7091007B2 (en) 1993-09-17 2006-08-15 Genetics Institute, Llc DNA molecules encoding BMP receptor proteins
US20070049688A1 (en) * 2005-07-19 2007-03-01 Antoni Kozlowski Method for preparing polymer maleimides
US20070048855A1 (en) * 2004-09-17 2007-03-01 Alejandra Gamez Variants and chemically-modified variants of phenylalanine ammonia-lyase
US7189392B1 (en) 1999-10-15 2007-03-13 Genetics Institute, Llc Injectable carrier formulations of hyaluronic acid derivatives for delivery of osteogenic proteins
US20070071764A1 (en) * 2005-04-22 2007-03-29 Sullivan John K Toxin peptide therapeutic agents
US20070092482A1 (en) * 2005-08-04 2007-04-26 Bossard Mary J Conjugates of a G-CSF moiety and a polymer
US7217691B2 (en) 1986-07-01 2007-05-15 Genetics Institute, Llc Methods of treatment of periodontal disease
US7226587B2 (en) 2001-06-01 2007-06-05 Wyeth Compositions and methods for systemic administration of sequences encoding bone morphogenetic proteins
US20070160658A1 (en) * 2005-10-20 2007-07-12 The Penn State Research Foundation Delivery system for diagnostic and therapeutic agents
US20070166278A1 (en) * 2004-04-15 2007-07-19 Veronese Francesco M Novel g-csf conjugates
US20080008695A1 (en) * 2006-06-12 2008-01-10 Vellard Michel C Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
US7323445B2 (en) 1999-02-01 2008-01-29 Genetics Institute, Llc Methods and compositions for healing and repair of articular cartilage
US20080076706A1 (en) * 1997-07-14 2008-03-27 Bolder Biotechnology, Inc. Derivatives of Growth Hormone and Related Proteins, and Methods of Use Thereof
US7408026B1 (en) * 1998-02-12 2008-08-05 The Scripps Research Institute Synthesis of proteins by native chemical ligation
US20080187955A1 (en) * 2001-10-10 2008-08-07 Neose Technologies, Inc. Erythropoietin: remodeling and glycoconjugation of erythropoietin
US7413753B2 (en) 2001-06-08 2008-08-19 Wyeth Calcium phosphate delivery vehicles for osteoinductive proteins
US20080200651A1 (en) * 2005-06-17 2008-08-21 Novo Nordisk Healthcare A/G Selective Reduction and Derivatization of Engineered Proteins Comprising at Least One Non-Native Cysteine
US20080213276A1 (en) * 2002-05-08 2008-09-04 Per Lindquist Modulation of neural stem cells and neural progenitor cells
US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
US20080255026A1 (en) * 2005-05-25 2008-10-16 Glycopegylated Factor 1X Glycopegylated Factor Ix
US20090047268A1 (en) * 2007-08-17 2009-02-19 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
US7495087B2 (en) 1997-07-14 2009-02-24 Bolder Biotechnology, Inc. Cysteine muteins in the C-D loop of human interleukin-11
US20090075873A1 (en) * 2002-11-21 2009-03-19 Antonio Cruz Gastrin compositions and formulations, and methods of use and preparation
US20090088559A1 (en) * 1999-07-07 2009-04-02 Torben Halkier Method for preparing modified polypeptides
WO2009046080A1 (en) 2007-10-01 2009-04-09 Pharmaessentia Corp. N-terminal modified interferon-alpha
US7531341B1 (en) 2006-06-12 2009-05-12 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
US20090163411A1 (en) * 2005-10-03 2009-06-25 Bolder Biotechnology, Inc. Long acting vegf inhibitors and methods of use
US20090162380A1 (en) * 2005-01-05 2009-06-25 Scott Glaser Multispecific binding molecules comprising connecting peptides
US20090281028A1 (en) * 2006-10-25 2009-11-12 Sullivan John K OSK1 peptide analogs and pharmaceutical compositions
US20090305967A1 (en) * 2005-08-19 2009-12-10 Novo Nordisk A/S Glycopegylated factor vii and factor viia
US7678885B2 (en) 1991-11-04 2010-03-16 Genetics Institute, Llc Recombinant bone morphogenetic protein heterodimers, compositions and methods of use
EP2186525A2 (en) 2002-12-31 2010-05-19 SYGNIS Bioscience GmbH & Co KG G-CSF for use in the treatment of Parkinson's disease
US20100143288A1 (en) * 2005-06-13 2010-06-10 Cj Cheiljedang Corporation Human granulocyte-colony stimulating factor isoforms
US7771755B2 (en) 2003-09-12 2010-08-10 Wyeth Injectable calcium phosphate solid rods and pastes for delivery of osteogenic proteins
US20100261872A1 (en) * 2001-10-10 2010-10-14 Neose Technologies, Inc. Factor VIII: remodeling and glycoconjugation of factor VIII
US20100330060A1 (en) * 2003-12-03 2010-12-30 Novo Nordisk A/S Glycopegylated factor ix
EP2295069A1 (en) 2004-06-30 2011-03-16 Sygnis Bioscience GmbH & Co. KG Treatment of neurological disorders with haematopoietic growth factors
US20110201022A1 (en) * 2008-07-30 2011-08-18 Biomarin Pharmaceutical Inc. Assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US8008453B2 (en) 2005-08-12 2011-08-30 Amgen Inc. Modified Fc molecules
US8053410B2 (en) 2002-06-21 2011-11-08 Novo Nordisk Health Care A/G Pegylated factor VII glycoforms
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates
EP2500032A1 (en) 2002-06-24 2012-09-19 Genentech, Inc. APO-2 ligand/trail variants and uses thereof
WO2012125973A2 (en) 2011-03-16 2012-09-20 Amgen Inc. Potent and selective inhibitors of nav1.3 and nav1.7
US8288126B2 (en) 1999-01-14 2012-10-16 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US8420779B2 (en) 2007-05-22 2013-04-16 Amgen Inc. Compositions and methods for producing bioactive fusion proteins
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
WO2014099984A1 (en) 2012-12-20 2014-06-26 Amgen Inc. Apj receptor agonists and uses thereof
US8791066B2 (en) 2004-07-13 2014-07-29 Novo Nordisk A/S Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1]
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
US8853161B2 (en) 2003-04-09 2014-10-07 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
WO2014165277A2 (en) 2013-03-12 2014-10-09 Amgen Inc. POTENT AND SELECTIVE INHIBITORS OF Nav1.7
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
WO2015191781A2 (en) 2014-06-10 2015-12-17 Amgen Inc. Apelin polypeptides
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
WO2017075173A2 (en) 2015-10-30 2017-05-04 Genentech, Inc. Anti-factor d antibodies and conjugates

Families Citing this family (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9107846D0 (en) * 1990-04-30 1991-05-29 Ici Plc Polypeptides
ES2113354T3 (en) * 1990-05-04 1998-05-01 American Cyanamid Co Stabilization somatotropins and other proteins by modification of cysteine ​​residues.
WO1992006116A1 (en) * 1990-09-28 1992-04-16 Ortho Pharmaceutical Corporation Hybrid growth factors
CA2070751A1 (en) * 1991-07-24 1993-01-25 John Edward Fitton Proteins
CA2124672A1 (en) * 1991-12-10 1993-06-24 Tse W. Chang Cytokines with an upaired cysteine residue and conjugates thereof
WO1993012145A1 (en) * 1991-12-19 1993-06-24 Baylor College Of Medicine Pva or peg conjugates of peptides for epitope-specific immunosuppression
US5578496A (en) * 1991-12-19 1996-11-26 Board Of Regents, Baylor College Of Medicine Detection of autoantibodies associated with the disease myasthenia gravis
US5772992A (en) * 1992-11-24 1998-06-30 G.D. Searle & Co. Compositions for co-administration of interleukin-3 mutants and other cytokines and hematopoietic factors
US6153183A (en) 1992-11-24 2000-11-28 G. D. Searle & Company Co-administration of interleukin-3 mutant polypeptides with CSF's or cytokines for multi-lineage hematopoietic cell production
US6361976B1 (en) 1992-11-24 2002-03-26 S. Christopher Bauer Co-administration of interleukin-3 mutant polypeptides with CSF'S for multi-lineage hematopoietic cell production
US6403076B1 (en) 1992-11-24 2002-06-11 S. Christopher Bauer Compositions for increasing hematopoiesis with interleukin-3 mutants
US5581476A (en) * 1993-01-28 1996-12-03 Amgen Inc. Computer-based methods and articles of manufacture for preparing G-CSF analogs
US5420019A (en) 1993-02-02 1995-05-30 Xoma Corporation Stable bactericidal/permeability-increasing protein muteins
US5501962A (en) * 1993-06-21 1996-03-26 G. D. Searle & Co. Interleuken-3 (IL-3) human/murine hybrid polypeptides and recombinant production of the same
US20030220233A1 (en) 1994-01-24 2003-11-27 Neorx Corporation Radiolabeled annexins
US5968477A (en) 1994-01-24 1999-10-19 Neorx Corporation Radiolabeled annexin conjugates with hexose and a chelator
JPH10500693A (en) * 1994-05-24 1998-01-20 アムジエン・ブルダー・インコーポレーテツド Modified insulin-like growth factor
RU2199347C2 (en) * 1996-08-02 2003-02-27 Орто-Макнейл Фармасьютикал, Инк. Polypeptides comprising a single covalently bound n-terminal water-soluble polymer
US6743422B1 (en) 1996-10-15 2004-06-01 Amgen, Inc. Keratinocyte growth factor-2 products
DE69824039D1 (en) 1997-12-08 2004-06-24 Lexigen Pharm Corp Heterodimeric fusion proteins for use for targeted immunotherapy and general immune excitation
US6583272B1 (en) * 1999-07-02 2003-06-24 Hoffmann-La Roche Inc. Erythropoietin conjugates
WO2001007081A1 (en) 1999-07-21 2001-02-01 Lexigen Pharmaceuticals Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
WO2001036489A3 (en) * 1999-11-12 2001-11-08 Merck Patent Gmbh Erythropoietin forms with improved properties
US6555660B2 (en) 2000-01-10 2003-04-29 Maxygen Holdings Ltd. G-CSF conjugates
US6646110B2 (en) 2000-01-10 2003-11-11 Maxygen Holdings Ltd. G-CSF polypeptides and conjugates
US6831158B2 (en) 2000-01-10 2004-12-14 Maxygen Holdings Ltd. G-CSF conjugates
CA2399832C (en) 2000-02-11 2011-09-20 Stephen D. Gillies Enhancing the circulating half-life of antibody-based fusion proteins
WO2001076639A3 (en) * 2000-04-06 2002-05-10 Rory Finn Chemically-modified myelopoietin conjugates
CN1318443C (en) * 2000-05-16 2007-05-30 博尔德生物技术公司 Methods for refolding proteins containing free cysteine residues
US20040038878A1 (en) 2000-08-04 2004-02-26 Masahiko Tanikawa Injectable protein formulations
JP5485489B2 (en) 2000-08-11 2014-05-07 中外製薬株式会社 Antibody-containing stabilized formulation
CA2420850A1 (en) 2000-09-01 2003-02-28 Chugai Seiyaku Kabushiki Kaisha Solution formulations having long-term stability
EP1319182A2 (en) 2000-09-08 2003-06-18 Massachusetts Institute Of Technology G-csf analog compositions and methods
US7118737B2 (en) 2000-09-08 2006-10-10 Amylin Pharmaceuticals, Inc. Polymer-modified synthetic proteins
DE60144188D1 (en) * 2000-09-08 2011-04-21 Amylin Pharmaceuticals Inc "pseudo"-native chemische ligation
CA2438652A1 (en) 2001-02-19 2002-09-06 Merck Patent Gesellschaft Mit Beschraenkter Haftung Method for identification of t-cell epitopes and use for preparing molecules with reeduced immunogenicity
KR20090010127A (en) 2001-03-07 2009-01-28 메르크 파텐트 게엠베하 Expression technology for proteins containing a hybrid isotype antibody moiety
US7332289B2 (en) 2001-03-09 2008-02-19 Chugai Seiyaku Kabushiki Kaisha Method of purifying protein
US6992174B2 (en) 2001-03-30 2006-01-31 Emd Lexigen Research Center Corp. Reducing the immunogenicity of fusion proteins
US7531358B2 (en) 2001-04-17 2009-05-12 Chugai Seiyaku Kabushiki Kaisha Method of quantifying surfactant
ES2361664T3 (en) 2001-05-03 2011-06-21 Merck Patent Gmbh Recombinant tumor specific antibody and its use.
RU2312677C9 (en) 2001-12-04 2008-03-27 Мерк Патент Гмбх Immunocytokines possessing modulated selectivity
WO2003055526A3 (en) * 2001-12-21 2003-12-11 Maxygen Aps Erythropoietin conjugates
DE10209821A1 (en) 2002-03-06 2003-09-25 Biotechnologie Ges Mittelhesse Coupling of proteins to a modified polysaccharide
JP4489591B2 (en) 2002-08-27 2010-06-23 中外製薬株式会社 Method for stabilizing a protein solution formulation
WO2005014655A3 (en) 2003-08-08 2005-06-16 Harald Conradt Conjugates of hydroxyalkyl starch and a protein
EP1561756B1 (en) 2002-09-11 2015-12-23 Chugai Seiyaku Kabushiki Kaisha Method of purifying protein
JP4494977B2 (en) 2002-12-17 2010-06-30 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung Mice that bind to gd2 14.18 antibody in the humanized antibody (h14.18) and il-2 fusion protein
CN1823088B (en) 2003-05-12 2011-04-13 阿费麦克斯公司 Novel peptides that bind to the erythropoietin receptor
US7084245B2 (en) 2003-05-12 2006-08-01 Affymax, Inc. Peptides that bind to the erythropoietin receptor
WO2004100997A3 (en) 2003-05-12 2005-05-19 Affymax Inc Spacer moiety for poly(ethylene glycol) -modified peptides
CA2525464A1 (en) 2003-05-12 2004-11-25 Qun Yin Novel poly(ethylene glycol) modified compounds and uses thereof
WO2005000360A3 (en) 2003-05-23 2005-07-28 Nektar Therapeutics Al Corp Peg derivatives having an amidocarbonate linkage
JP2007537986A (en) 2003-05-30 2007-12-27 セントカー・インコーポレーテツド Formation of new erythropoietin complexes using transglutaminase
ES2405209T3 (en) * 2003-08-07 2013-05-30 Zymogenetics, Inc. homogeneous preparations of IL-29
US7220407B2 (en) 2003-10-27 2007-05-22 Amgen Inc. G-CSF therapy as an adjunct to reperfusion therapy in the treatment of acute myocardial infarction
KR101146160B1 (en) * 2004-06-30 2012-07-16 넥타르 테라퓨틱스 Polymer-factor ix moiety conjugates
WO2006055260A3 (en) 2004-11-05 2006-07-27 Univ Northwestern Use of scf and g-csf in the treatment of cerebral ischemia and neurological disorders
KR20120136413A (en) 2004-11-12 2012-12-18 바이엘 헬스케어 엘엘씨 Site-directed modification of fviii
WO2006089228A3 (en) 2005-02-16 2007-06-14 Mary J Bossard Conjugates of an epo moiety and a polymer
CN100381420C (en) * 2005-03-23 2008-04-16 于勇海 N-acetyl cysteine derivatives and use thereof
US8273339B2 (en) 2005-04-12 2012-09-25 Nektar Therapeutics Polymer-based compositions and conjugates of antimicrobial agents
US7919461B2 (en) 2005-06-03 2011-04-05 Affymax, Inc. Erythropoietin receptor peptide formulations and uses
US7550433B2 (en) 2005-06-03 2009-06-23 Affymax, Inc. Erythropoietin receptor peptide formulations and uses
US8324159B2 (en) 2005-06-03 2012-12-04 Affymax, Inc. Erythropoietin receptor peptide formulations and uses
RU2008145084A (en) * 2006-05-24 2010-06-27 Ново Нордиск Хелс Кеа Аг (Ch) Analogs factor ix, having a prolonged half-life in vivo
US8765924B2 (en) 2006-08-04 2014-07-01 Prolong Pharmaceuticals, Inc. Modified erythropoietin
CA2661054A1 (en) 2006-08-22 2008-02-28 Chugai Seiyaku Kabushiki Kaisha Prophylactic and/or therapeutic agents for peripheral neuropathy
WO2008076933A3 (en) 2006-12-14 2008-12-18 Bolder Biotechnology Inc Long acting proteins and peptides and methods of making and using the same
US8389482B2 (en) 2007-01-30 2013-03-05 New York University Short peptides useful for treatment of ischemia/reperfusion injury and other tissue damage conditions associated with nitric oxide and its reactive species
EP2118127A4 (en) 2007-01-31 2010-12-01 Affymax Inc Nitrogen-based linkers for attaching modifying groups to polypeptides and other macromolecules
JP5763882B2 (en) 2007-03-09 2015-08-12 鈴木 宏志 Transplanted organs protection agent
CA2697265A1 (en) 2007-08-09 2009-02-19 Genzyme Corporation Method of treating autoimmune disease with mesenchymal stem cells
US8758761B2 (en) 2007-09-30 2014-06-24 University Of Florida Research Foundation, Inc. Combination therapies for treating type 1 diabetes
EP3042922B1 (en) 2008-01-11 2017-07-19 Serina Therapeutics, Inc. Multifunctional forms of polyoxazoline copolymers and drug compositions comprising the same
US8101706B2 (en) 2008-01-11 2012-01-24 Serina Therapeutics, Inc. Multifunctional forms of polyoxazoline copolymers and drug compositions comprising the same
WO2010080720A3 (en) 2009-01-12 2010-08-26 Nektar Therapeutics Conjugates of a lysosomal enzyme moiety and a water soluble polymer
CA2791361A1 (en) 2010-03-04 2011-09-09 Pfenex Inc. Method for producing soluble recombinant interferon protein without denaturing
EP2552949B1 (en) 2010-04-01 2016-08-17 Pfenex Inc. Methods for g-csf production in a pseudomonas host cell
CA2816722A1 (en) 2010-11-12 2012-05-18 Nektar Therapeutics Conjugates of an il-2 moiety and a polymer
WO2013020079A3 (en) 2011-08-04 2013-04-18 Nektar Therapeutics Conjugates of an il-11 moiety and a polymer

Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3960830A (en) * 1973-12-06 1976-06-01 Hoechst Aktiengesellschaft Polyalkylene glycols used for the preparation of peptides
US4002531A (en) * 1976-01-22 1977-01-11 Pierce Chemical Company Modifying enzymes with polyethylene glycol and product produced thereby
GB1469472A (en) * 1973-07-20 1977-04-06 Research Corp Non-immunogenic polypeptides and processes for preparing same
US4055635A (en) * 1973-07-05 1977-10-25 Beecham Group Limited Fibrinolytic compositions
US4088538A (en) * 1975-05-30 1978-05-09 Battelle Memorial Institute Reversibly precipitable immobilized enzyme complex and a method for its use
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4261973A (en) * 1976-08-17 1981-04-14 Pharmacia Ab Allergen-containing substances
US4301144A (en) * 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4379086A (en) * 1981-06-09 1983-04-05 Fujizoki Pharmaceutical Co., Ltd. Method of preparing immunoglobulin suitable for intravenous administration using PEG
US4412989A (en) * 1981-06-10 1983-11-01 Ajinomoto Company Incorporated Oxygen carrier
US4415665A (en) * 1980-12-12 1983-11-15 Pharmacia Fine Chemicals Ab Method of covalently binding biologically active organic substances to polymeric substances
US4495285A (en) * 1981-10-30 1985-01-22 Kimihiro Shimizu Plasminogen activator derivatives
US4496689A (en) * 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
EP0154316A2 (en) * 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Chemically modified lymphokine and production thereof
WO1986004145A1 (en) * 1984-12-31 1986-07-17 University Of New Mexico Protein modification with peg
US4609546A (en) * 1982-06-24 1986-09-02 Japan Chemical Research Co., Ltd. Long-acting composition
WO1987000056A1 (en) * 1985-06-26 1987-01-15 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4640835A (en) * 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4703039A (en) * 1984-04-10 1987-10-27 New England Deaconess Hospital Corporation Method of producing biologically active molecules having extended life time
US4737462A (en) * 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4791192A (en) * 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4818769A (en) * 1985-09-20 1989-04-04 Cetus Corporation Method of controlling stress-related disease in livestock by administration of human IL-2
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
US4877729A (en) * 1986-07-14 1989-10-31 Genetics Institute, Inc. Recombinant DNA encoding novel family of primate hematopoietic growth factors
US4898824A (en) * 1986-12-09 1990-02-06 Miles Inc. Crosslinked polyacrylamide-sulfhydryl polymer for immobilization of biologically active substances
US4902502A (en) * 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
US4959455A (en) * 1986-07-14 1990-09-25 Genetics Institute, Inc. Primate hematopoietic growth factors IL-3 and pharmaceutical compositions
EP0254172B1 (en) * 1986-07-24 1991-11-06 Miles Inc. Enzyme-labeled antibody reagent with polyalkyleneglycol linking group

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5206344A (en) * 1985-06-26 1993-04-27 Cetus Oncology Corporation Interleukin-2 muteins and polymer conjugation thereof
JP2514950B2 (en) * 1986-03-10 1996-07-10 エフ・ホフマン―ラ ロシユ アーゲー Chemically modified proteins, their production and intermediates
CA1283046C (en) * 1986-05-29 1991-04-16 Nandini Katre Tumor necrosis factor formulation
WO1988001297A1 (en) * 1986-08-11 1988-02-25 Cetus Corporation Expression of g-csf and muteins thereof
US4835260A (en) * 1987-03-20 1989-05-30 Genetics Institute, Inc. Erythropoietin composition
JPH0796558B2 (en) * 1988-03-31 1995-10-18 協和醗酵工業株式会社 Modified polypeptide
EP0355460B1 (en) * 1988-08-24 2000-12-27 American Cyanamid Company Stabilization of somatotropins by modification of cysteine residues utilizing site directed mutagenesis or chemical derivatization
ES2113354T3 (en) * 1990-05-04 1998-05-01 American Cyanamid Co Stabilization somatotropins and other proteins by modification of cysteine ​​residues.
CA2106079C (en) * 1991-03-15 2000-04-25 Robert C. Thompson Pegylation of polypeptides
CA2149048A1 (en) * 1992-11-25 1994-06-09 George N. Cox Modified insulin-like growth factors

Patent Citations (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4055635A (en) * 1973-07-05 1977-10-25 Beecham Group Limited Fibrinolytic compositions
GB1469472A (en) * 1973-07-20 1977-04-06 Research Corp Non-immunogenic polypeptides and processes for preparing same
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US3960830A (en) * 1973-12-06 1976-06-01 Hoechst Aktiengesellschaft Polyalkylene glycols used for the preparation of peptides
US4088538A (en) * 1975-05-30 1978-05-09 Battelle Memorial Institute Reversibly precipitable immobilized enzyme complex and a method for its use
US4002531A (en) * 1976-01-22 1977-01-11 Pierce Chemical Company Modifying enzymes with polyethylene glycol and product produced thereby
US4261973A (en) * 1976-08-17 1981-04-14 Pharmacia Ab Allergen-containing substances
US4301144A (en) * 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4415665A (en) * 1980-12-12 1983-11-15 Pharmacia Fine Chemicals Ab Method of covalently binding biologically active organic substances to polymeric substances
US4379086A (en) * 1981-06-09 1983-04-05 Fujizoki Pharmaceutical Co., Ltd. Method of preparing immunoglobulin suitable for intravenous administration using PEG
US4412989A (en) * 1981-06-10 1983-11-01 Ajinomoto Company Incorporated Oxygen carrier
US4495285A (en) * 1981-10-30 1985-01-22 Kimihiro Shimizu Plasminogen activator derivatives
US4495285B1 (en) * 1981-10-30 1986-09-23 Nippon Chemiphar Co
US4640835A (en) * 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4609546A (en) * 1982-06-24 1986-09-02 Japan Chemical Research Co., Ltd. Long-acting composition
US4737462A (en) * 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
EP0147761A2 (en) * 1983-12-27 1985-07-10 Miles Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4496689A (en) * 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
EP0154316A2 (en) * 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Chemically modified lymphokine and production thereof
US4703039A (en) * 1984-04-10 1987-10-27 New England Deaconess Hospital Corporation Method of producing biologically active molecules having extended life time
WO1986004145A1 (en) * 1984-12-31 1986-07-17 University Of New Mexico Protein modification with peg
WO1987000056A1 (en) * 1985-06-26 1987-01-15 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4766106A (en) * 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
US4818769A (en) * 1985-09-20 1989-04-04 Cetus Corporation Method of controlling stress-related disease in livestock by administration of human IL-2
US4791192A (en) * 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4877729A (en) * 1986-07-14 1989-10-31 Genetics Institute, Inc. Recombinant DNA encoding novel family of primate hematopoietic growth factors
US4959455A (en) * 1986-07-14 1990-09-25 Genetics Institute, Inc. Primate hematopoietic growth factors IL-3 and pharmaceutical compositions
EP0254172B1 (en) * 1986-07-24 1991-11-06 Miles Inc. Enzyme-labeled antibody reagent with polyalkyleneglycol linking group
US4898824A (en) * 1986-12-09 1990-02-06 Miles Inc. Crosslinked polyacrylamide-sulfhydryl polymer for immobilization of biologically active substances
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
US4902502A (en) * 1989-01-23 1990-02-20 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
Abuchowski et al., Cancer Biochem Biophys 7:175 86 (1984). *
Abuchowski et al., Cancer Biochem Biophys 7:175-86 (1984).
Boccu et al, Z. Naturforsch. 38c, 1983, pp. 94 99. *
Boccu et al, Z. Naturforsch. 38c, 1983, pp. 94-99.
Carlsson et al., Biochem. J. 173: 723 37 (1978). *
Carlsson et al., Biochem. J. 173: 723-37 (1978).
Clark Lewis et al, PNAS 85, pp. 7897 7901, Nov. 1988. *
Clark-Lewis et al, PNAS 85, pp. 7897-7901, Nov. 1988.
Dunbar et al, Science, 1989, pp. 1493 1496. *
Dunbar et al, Science, 1989, pp. 1493-1496.
Furukawa et al., FEBS letters 121:239 42 (1980). *
Furukawa et al., FEBS letters 121:239-42 (1980).
Inada et al., Biochem and Biophys Res Comm 122:848 50 (1984). *
Inada et al., Biochem and Biophys Res Comm 122:848-50 (1984).
Katre, et al., Proc. Natl. Acad. Sci. USA 84:1487 91 (1987). *
Katre, et al., Proc. Natl. Acad. Sci. USA 84:1487-91 (1987).
Takahashi et al., Biochem and Biophys Res Comm 121:261 65. *
Takahashi et al., Biochem and Biophys Res Comm 121:261-65.
Takakura et al., J. Pharm. Sci. 87:117 21 (1989). *
Takakura et al., J. Pharm. Sci. 87:117-21 (1989).
Wingfield et al., Eur. J. Biochem. 179:565 71 (1989). *
Wingfield et al., Eur. J. Biochem. 179:565-71 (1989).

Cited By (306)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5718893A (en) * 1984-04-15 1998-02-17 Foster; Preston F. Use of G-CSF to reduce acute rejection
US5359035A (en) * 1985-12-21 1994-10-25 Hoechst Aktiengesellschaft Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF)
US7300772B2 (en) 1986-07-01 2007-11-27 Genetics Institute, Llc BMP products
US7217691B2 (en) 1986-07-01 2007-05-15 Genetics Institute, Llc Methods of treatment of periodontal disease
US5880255A (en) * 1988-10-20 1999-03-09 Polymasc Pharmaceuticals Plc Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct of PEG and granulocyte-macrophage colony stimulating factor
US6384195B1 (en) * 1988-10-20 2002-05-07 Polymasc Pharmaceuticals Plc. Process for fractionating polyethylene glycol (PEG) —protein adducts and an adduct of PEG and granulocyt-macrophage colony stimulating factor
US5824778A (en) * 1988-12-22 1998-10-20 Kirin-Amgen, Inc. Chemically-modified G-CSF
US20020177688A1 (en) * 1988-12-22 2002-11-28 Kirin-Amgen, Inc., Chemically-modified G-CSF
US20030204057A1 (en) * 1988-12-22 2003-10-30 Kirin-Amgen Chemically modified G-CSF
US20070219357A1 (en) * 1988-12-22 2007-09-20 Kirin-Amgen, Inc. Chemically modified G-CSF
US6143866A (en) * 1989-07-18 2000-11-07 Amgen, Inc. Tumor necrosis factor (TNF) inhibitor and method for obtaining the same
US6541620B1 (en) 1989-07-18 2003-04-01 Angen Inc. Nucleic acids encoding TNF inhibitor and method of production
US5945397A (en) * 1989-09-05 1999-08-31 Immunex Corporation Purified p75 (type II) tumor necrosis factor receptor polypeptides
US20030190304A1 (en) * 1990-04-06 2003-10-09 Amgen Inc. Pegylation reagents and compounds formed therewith
US6552170B1 (en) * 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
US6010999A (en) * 1990-05-04 2000-01-04 American Cyanamid Company Stabilization of fibroblast growth factors by modification of cysteine residues
US5951972A (en) * 1990-05-04 1999-09-14 American Cyanamid Company Stabilization of somatotropins and other proteins by modification of cysteine residues
US5766897A (en) * 1990-06-21 1998-06-16 Incyte Pharmaceuticals, Inc. Cysteine-pegylated proteins
US5883230A (en) * 1990-08-29 1999-03-16 Genetics Institute, Inc. Multidomain hematopoiesis stimulators
US5856138A (en) * 1991-08-07 1999-01-05 Takeda Chemical Industries, Ltd. Human parathyroid hormone muteins and production thereof
US7678885B2 (en) 1991-11-04 2010-03-16 Genetics Institute, Llc Recombinant bone morphogenetic protein heterodimers, compositions and methods of use
US5705362A (en) * 1992-05-25 1998-01-06 Gist-Brocades, N.V. Modified signal sequences
US5677149A (en) * 1992-11-24 1997-10-14 G.D. Searle & Co., Interleukin-3 (IL-3) mutant polypeptides and their recombinant production
US6479261B1 (en) 1992-11-24 2002-11-12 Pharmacia Corporation Methods of using interleukin-3 (IL-3) mutant polypeptides for ex-vivo expansion of hematopoietic stem cells
US6458931B1 (en) 1992-11-24 2002-10-01 S. Christopher Bauer Interleukin-3 (IL-3) multiple mutation polypeptides
US20050244372A1 (en) * 1992-11-24 2005-11-03 Bauer S C Interleukin-3 (IL-3) multiple mutation polypeptides
US6074639A (en) * 1992-11-24 2000-06-13 G. D. Searle & Co. Ex vivo expansion of hematopoietic cells using interleukin-3 (IL-3) variant fusion proteins
US6440407B1 (en) 1992-11-24 2002-08-27 G. D. Searle Methods of ex-vivo expansion of hematopoietic cells using interleukin-3 (IL-3) multiple mutation polypeptides
US5817486A (en) * 1992-11-24 1998-10-06 G. D. Searle & Co. Recombinant human interleukin-3 (IL-3) multiple mutation polypeptides
US6051217A (en) * 1992-11-24 2000-04-18 G. D. Searle & Co. Therapeutic uses of interleukin-3 (IL-3) multiple mutation polypeptides
US5604116A (en) * 1992-11-24 1997-02-18 G. D. Searle & Co. Interleukin-3 (IL-3) multiple mutation polypeptides, recombinant production of the same, and corresponding therapeutic methods
WO1994013316A1 (en) * 1992-12-16 1994-06-23 Board Of Regents, The University Of Texas System Potent and specific chemically-conjugated immunotoxins
US7091007B2 (en) 1993-09-17 2006-08-15 Genetics Institute, Llc DNA molecules encoding BMP receptor proteins
WO1995011987A1 (en) * 1993-10-29 1995-05-04 Incyte Pharmaceuticals, Inc. Chimeric proteins including protease nexin-1 variants
US5932427A (en) * 1993-11-10 1999-08-03 Henry M. Jackson Foundation For The Advancement Of Military Medicine In vitro assay system for identifying compositions useful for stimulating B cells
US6984623B2 (en) 1993-12-07 2006-01-10 Genetics, Institute Institute, LLC. Tendon-inducing compositions
US7365052B2 (en) 1993-12-07 2008-04-29 Genetics Institute, Llc. Tendon-inducing methods
US5536495A (en) * 1994-04-15 1996-07-16 Foster; Preston F. Use of G-CSF to reduce acute rejection
US20030053982A1 (en) * 1994-09-26 2003-03-20 Kinstler Olaf B. N-terminally chemically modified protein compositions and methods
EP0785276A1 (en) * 1994-09-29 1997-07-23 Ajinomoto Co., Inc. Modification of peptide and protein
US8258262B2 (en) 1994-10-12 2012-09-04 Amgen Inc. N-terminally chemically modified protein compositions and methods
US6956027B2 (en) 1994-10-12 2005-10-18 Amgen Inc. N-terminally chemically modified protein compositions and methods
US20100310510A1 (en) * 1994-10-12 2010-12-09 Amgen Inc. N-terminally chemically modified protein compositions and methods
US20040181035A1 (en) * 1994-10-12 2004-09-16 Amgen, Inc. N-terminally chemically modified protein compositions and methods
US7090835B2 (en) 1994-10-12 2006-08-15 Amgen, Inc. N-terminally chemically modified protein compositions and methods
US20060233746A1 (en) * 1994-10-12 2006-10-19 Amgen Inc. N-terminally chemically modified protein compositions and methods
US7662933B2 (en) 1994-10-12 2010-02-16 Amgen Inc. N-terminally chemically modified protein compositions and methods
US20030096400A1 (en) * 1994-10-12 2003-05-22 Amgen, Inc. N-terminally chemically modified protein compositions and methods
US5846770A (en) * 1994-11-22 1998-12-08 Genetics Institute, Inc. DNA molecules encoding human chordin
US5986056A (en) * 1994-11-22 1999-11-16 Lavallie; Edward R. Chordin compositions
US5747027A (en) * 1995-04-07 1998-05-05 The Regents Of The University Of California BH55 hyaluronidase
US5827721A (en) * 1995-04-07 1998-10-27 The Regents Of The University Of California BH55 hyaluronidase
US6017523A (en) * 1995-06-06 2000-01-25 G.D. Searle & Co. Therapeutic methods employing mutant human interleukin-3 (IL-3) polypeptides
US5830761A (en) * 1995-06-07 1998-11-03 Genetics Institute, Inc. Medium and methods for culturing mammalian cho cells
US5976783A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Bis-maleimido cross-linking agents
US5976857A (en) * 1996-01-26 1999-11-02 Boehringer Mannheim Corporation Cross-linked polypeptide fragments of β-galactosidase
US5763196A (en) * 1996-01-26 1998-06-09 Boehringer Mannheim Corporation Assays using cross-linked polypeptide fragments of β-galactosidase
US6989147B2 (en) 1996-07-09 2006-01-24 Amgen Inc. Truncated soluble tumor necrosis factor type-I and type-II receptors
US20050250696A1 (en) * 1996-07-09 2005-11-10 Fisher Eric F Truncated soluble tumor necrosis factor type-I and type-II receptors
US7732587B2 (en) 1996-07-09 2010-06-08 Amgen Inc. Nucleic acids encoding truncated soluble tumor necrosis factor
US5952226A (en) * 1996-11-05 1999-09-14 Modex Therapeutiques Hypoxia responsive EPO producing cells
US6306820B1 (en) 1996-12-06 2001-10-23 Amgen Inc. Combination therapy using a TNF binding protein for treating TNF-mediated diseases
US6323006B1 (en) * 1996-12-18 2001-11-27 Universidade Federal De Minas Gerais Recombinant human beta-CIS interferon
US6870033B1 (en) 1997-02-21 2005-03-22 Genentech, Inc. Antibody fragment-polymer conjugates and humanized anti-IL-8 monoclonal antibodies
US7122636B1 (en) 1997-02-21 2006-10-17 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US5935852A (en) * 1997-07-03 1999-08-10 Genetics Institute, Inc. DNA molecules encoding mammalian cerberus-like proteins
US7148333B2 (en) 1997-07-14 2006-12-12 Bolder Biotechnology, Inc. Cysteine variants of granulocyte-macrophage colony-stimulating factor
US20080076706A1 (en) * 1997-07-14 2008-03-27 Bolder Biotechnology, Inc. Derivatives of Growth Hormone and Related Proteins, and Methods of Use Thereof
US7345149B2 (en) 1997-07-14 2008-03-18 Bolder Biotechnology, Inc. Cysteine substitution variants of beta interferon
US7345154B2 (en) 1997-07-14 2008-03-18 Bolder Biotechnology, Inc. Cysteine variants of erythropoietin
US20040175800A1 (en) * 1997-07-14 2004-09-09 Bolder Biotechnology, Inc. Cysteine variants of granulocyte colony-stimulating factor
US20040175356A1 (en) * 1997-07-14 2004-09-09 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11
US20030171284A1 (en) * 1997-07-14 2003-09-11 Cox George N. Derivatives of growth hormone and related proteins, and methods of use thereof
US20030166865A1 (en) * 1997-07-14 2003-09-04 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11
US8148500B2 (en) 1997-07-14 2012-04-03 Bolder Biotechnology, Inc. Method of preparing cysteine mutants of human recombinant GM-CSF
US20040214287A1 (en) * 1997-07-14 2004-10-28 Bolder Biotechnology, Inc. Cysteine variants of granulocyte-macrophage colony-stimulating factor
US8133480B2 (en) 1997-07-14 2012-03-13 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11
US7994124B2 (en) 1997-07-14 2011-08-09 Bolder Biotechnology, Inc. Methods of use of G-CSF cysteine muteins
US20110172151A1 (en) * 1997-07-14 2011-07-14 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins, and methods of use thereof
US20040230040A1 (en) * 1997-07-14 2004-11-18 Bolder Biotechnology, Inc. Cysteine variants of alpha interferon-2
US7964184B2 (en) 1997-07-14 2011-06-21 Bolder Biotechnology, Inc. Cysteine variants of interferon-gamma
US20040265269A1 (en) * 1997-07-14 2004-12-30 Bolder Biotechnology, Inc. Cysteine variants of granulocyte colony-stimulating factor
US8455434B2 (en) 1997-07-14 2013-06-04 Bolder Biotechnology, Inc. Methods of use for cysteine variants of human G-CSF
US20050058621A1 (en) * 1997-07-14 2005-03-17 Cox George N. Derivatives of growth hormone and related proteins, and methods of use thereof
US6608183B1 (en) * 1997-07-14 2003-08-19 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
EP1881005A1 (en) 1997-07-14 2008-01-23 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US20050096461A1 (en) * 1997-07-14 2005-05-05 Bolder Biotechnology, Inc. Cysteine variants of erythropoietin
US20050107591A1 (en) * 1997-07-14 2005-05-19 Bolder Biotechnology, Inc. Cysteine variants of erythropoietin
US20080317713A1 (en) * 1997-07-14 2008-12-25 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US7314921B2 (en) 1997-07-14 2008-01-01 Bolder Biotechnology, Inc. Cysteine variants of erythropoietin
US7309781B2 (en) * 1997-07-14 2007-12-18 Bolder Biotechnology, Inc. Cysteine variants of granulocyte colony-stimulating factor
US20070253929A1 (en) * 1997-07-14 2007-11-01 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins, and methods of use thereof
US7495087B2 (en) 1997-07-14 2009-02-24 Bolder Biotechnology, Inc. Cysteine muteins in the C-D loop of human interleukin-11
US7270809B2 (en) * 1997-07-14 2007-09-18 Bolder Biotechnology, Inc. Cysteine variants of alpha interferon-2
US7824669B2 (en) 1997-07-14 2010-11-02 Bolder Biotechnology, Inc. In vivo stimulation of peripheral blood progenitor cells by granulocyte-macrophage colony stimulating factor (GM-CSF) cysteine muteins and their PEGylated variants
US20050214254A1 (en) * 1997-07-14 2005-09-29 Bolder Biotechnology, Inc. Cysteine variants of beta interferon
US20050227330A1 (en) * 1997-07-14 2005-10-13 Bolder Biotechnology, Inc. Cysteine variants of beta interferon
US8618256B2 (en) 1997-07-14 2013-12-31 Bolder Biotechnology Cysteine variants of interferon gamma
US7253267B2 (en) 1997-07-14 2007-08-07 Bolder Biotechnology Inc. Cysteine variants of interleukin-11
US20090060863A1 (en) * 1997-07-14 2009-03-05 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US8748392B2 (en) 1997-07-14 2014-06-10 Bolder Biotechnology Inc. Methods of treatment using cysteine variants of interleukin-11
US7795396B2 (en) 1997-07-14 2010-09-14 Bolder Biotechnology, Inc. COOH-terminally added cysteine variant of the beta interferon
US8859497B2 (en) 1997-07-14 2014-10-14 Bolder Biotechnology, Inc. Method of treatment using cysteine mutants of beta interferon
US7232885B2 (en) * 1997-07-14 2007-06-19 Bolder Biotechnology, Inc. Termini cysteine-added variants of granulocyte colony stimulating factor
US7732572B2 (en) 1997-07-14 2010-06-08 Bolder Biotechnology, Inc. Cysteine variants of alpha interferon-2
US7560101B2 (en) 1997-07-14 2009-07-14 Bolder Biotechnology, Inc. Method of treatment of an infection in an animal with GM-CSF cysteine muteins in the proximal region to helix A
US7959909B2 (en) 1997-07-14 2011-06-14 Bolder Biotechnology, Inc. Cysteine variants of interferon gamma
US7214779B2 (en) * 1997-07-14 2007-05-08 Bolder Biotechnology Inc. Termini cysteine-added variants of granulocyte-macrophage colony stimulating factor
US20090281281A1 (en) * 1997-07-14 2009-11-12 Bolder Biotechnology, Inc. Cysteine variants of interleukin-11 and methods of use thereof
US20080219950A1 (en) * 1997-07-14 2008-09-11 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US7153943B2 (en) 1997-07-14 2006-12-26 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins, and methods of use thereof
US9637530B2 (en) 1997-07-14 2017-05-02 Bolder Biotechnology, Inc. Methods of preparing cysteine variants of human recombinant IL-11
WO1999003887A1 (en) * 1997-07-14 1999-01-28 Bolder Biotechnology, Inc. Derivatives of growth hormone and related proteins
US6770448B2 (en) 1997-08-14 2004-08-03 The Regents Of The University Of California Fluorescent amyloid Aβ peptides and uses thereof
US6600017B1 (en) * 1997-08-14 2003-07-29 The Regents Of The University Of California Fluorescent amyloid Aβ peptides and uses thereof
US6458355B1 (en) 1998-01-22 2002-10-01 Genentech, Inc. Methods of treating inflammatory disease with anti-IL-8 antibody fragment-polymer conjugates
US6468532B1 (en) 1998-01-22 2002-10-22 Genentech, Inc. Methods of treating inflammatory diseases with anti-IL-8 antibody fragment-polymer conjugates
US7005504B2 (en) 1998-01-22 2006-02-28 Genentech, Inc. Antibody fragment-peg conjugates
US20030021790A1 (en) * 1998-01-22 2003-01-30 Genentech, Inc. Antibody fragment-polymer conjugates and humanized anti-IL-8 monoclonal antibodies
US7408026B1 (en) * 1998-02-12 2008-08-05 The Scripps Research Institute Synthesis of proteins by native chemical ligation
WO1999055377A3 (en) * 1998-04-28 1999-12-29 Applied Research Systems Polyol-ifn-beta conjugates
WO1999055377A2 (en) * 1998-04-28 1999-11-04 Applied Research Systems Ars Holding N.V. Polyol-ifn-beta conjugates
US7700314B2 (en) 1998-04-28 2010-04-20 Merck Serono Sa Method for producing polyol-IFN-β conjugate
US20070141620A1 (en) * 1998-04-28 2007-06-21 Applied Research Systems Ars Holding N.V. Polyol-ifn-beta conjugate and composition containing same
US7357925B2 (en) 1998-04-28 2008-04-15 Laboratoires Seronosa Method for treating disorders and diseases treatable with human fibroblast interferon
US6638500B1 (en) 1998-04-28 2003-10-28 Applied Research Systems Ars Holding N.V. Polyol-IFN-βconjugates modified at Cys-17 and composition containing same
US6420339B1 (en) * 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
US7186810B2 (en) 1998-10-23 2007-03-06 Amgen Inc. Modified peptides as therapeutic agents
US20040087778A1 (en) * 1998-10-23 2004-05-06 Amgen Inc. Modified peptides as therapeutic agents
WO2000042175A1 (en) 1999-01-14 2000-07-20 Bolder Biotechnology Inc. Methods for making proteins containing free cysteine residues
US7399839B2 (en) 1999-01-14 2008-07-15 Bolder Biotechnology, Inc. Monopegylated growth hormone proteins
US20050187160A1 (en) * 1999-01-14 2005-08-25 Bolder Biotechnology Inc. Monopegylated growth hormone proteins
US8288126B2 (en) 1999-01-14 2012-10-16 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US8957023B2 (en) 1999-01-14 2015-02-17 Bolder Biotechnology Inc. Methods for making proteins containing free cysteine residues
US20050026834A1 (en) * 1999-01-14 2005-02-03 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US7629314B2 (en) 1999-01-14 2009-12-08 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
EP2305804A1 (en) 1999-01-14 2011-04-06 Bolder Biotechnology, Inc. MonoPEGylated human growth hormone
US7947655B2 (en) 1999-01-14 2011-05-24 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US20100048872A1 (en) * 1999-01-14 2010-02-25 Bolder Biotechnology, Inc. Methods for making proteins containing free cysteine residues
US20110104185A1 (en) * 1999-01-21 2011-05-05 Vanessa Hsei Antibody fragment-polymer conjugates and uses of same
US8147830B2 (en) 1999-01-21 2012-04-03 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US7842789B2 (en) 1999-01-21 2010-11-30 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US8652468B2 (en) 1999-01-21 2014-02-18 Genentech, Inc. Methods of binding TNF-α using anti-TNF-α antibody fragment-polymer conjugates
US20090098147A1 (en) * 1999-01-21 2009-04-16 Vanessa Hsei Antibody fragment-polymer conjugates and uses of same
US7214776B2 (en) 1999-01-21 2007-05-08 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US20060083683A1 (en) * 1999-01-21 2006-04-20 Vanessa Hsei Antibody fragment-polymer conjugates and uses of same
US7507405B2 (en) 1999-01-21 2009-03-24 Genentech, Inc. Antibody fragment-polymer conjugates and uses of same
US20070048219A1 (en) * 1999-01-21 2007-03-01 Vanessa Hsei Antibody fragment-polymer conjugates and uses of same
US20030130193A1 (en) * 1999-01-29 2003-07-10 Bailon Pascal Sebastian GCSF conjugates
US20050196378A1 (en) * 1999-01-29 2005-09-08 Bailon Pascal S. G-CSF conjugates
US20070219356A1 (en) * 1999-01-29 2007-09-20 Bailon Pascal S G-CSF conjugates
US20080287659A1 (en) * 1999-01-29 2008-11-20 Amgen Inc. G-CSF Conjugates
US7323445B2 (en) 1999-02-01 2008-01-29 Genetics Institute, Llc Methods and compositions for healing and repair of articular cartilage
US20090088559A1 (en) * 1999-07-07 2009-04-02 Torben Halkier Method for preparing modified polypeptides
US7189392B1 (en) 1999-10-15 2007-03-13 Genetics Institute, Llc Injectable carrier formulations of hyaluronic acid derivatives for delivery of osteogenic proteins
US6911197B2 (en) 2000-02-22 2005-06-28 Nektar Therapeutics Al, Corporation N-maleimidyl polymer derivatives
US8992902B2 (en) 2000-02-22 2015-03-31 Nektar Therapeutics N-maleimidyl polymer derivatives
US9169350B2 (en) 2000-02-22 2015-10-27 Nektar Therapeutics N-maleimidyl polymer derivatives
US8765111B2 (en) 2000-02-22 2014-07-01 Nektar Therapeutics N-maleimidyl polymer derivatives
US20060013797A1 (en) * 2000-02-22 2006-01-19 Xiaoming Shen N-maleimidyl polymer derivatives
US6602498B2 (en) 2000-02-22 2003-08-05 Shearwater Corporation N-maleimidyl polymer derivatives
US8454946B2 (en) 2000-02-22 2013-06-04 Nektar Therapeutics N-maleimidyl polymer derivatives
US9493609B2 (en) 2000-02-22 2016-11-15 Nektar Therapeutics N-maleimidyl polymer derivatives
US20040109841A1 (en) * 2000-02-22 2004-06-10 Nektar Therapeutics Al, Corporation N-maleimidyl polymer derivatives
US9828465B2 (en) 2000-02-22 2017-11-28 Nektar Therapeutics N-maleimidyl polymer derivatives
US6818435B2 (en) 2000-05-15 2004-11-16 Tecan Trading Ag Microfluidics devices and methods for performing cell based assays
US7235638B2 (en) 2001-03-22 2007-06-26 Novo Nordisk Healthcare A/G Coagulation factor VII derivatives
US20030044908A1 (en) * 2001-03-22 2003-03-06 Egon Persson Coagulation factor VII derivatives
WO2002077218A1 (en) 2001-03-22 2002-10-03 Novo Nordisk Health Care Ag Coagulation factor vii derivatives
US20030017883A1 (en) * 2001-05-28 2003-01-23 Hiroaki Yoshiike Swing analyzing device
US7226587B2 (en) 2001-06-01 2007-06-05 Wyeth Compositions and methods for systemic administration of sequences encoding bone morphogenetic proteins
US7413753B2 (en) 2001-06-08 2008-08-19 Wyeth Calcium phosphate delivery vehicles for osteoinductive proteins
EP2348043A1 (en) 2001-10-02 2011-07-27 Genentech, Inc. APO-2 ligand variants and uses thereof
US20090137476A1 (en) * 2001-10-02 2009-05-28 Genentech, Inc. Apo-2 ligand variants and uses thereof
US20040186051A1 (en) * 2001-10-02 2004-09-23 Kelley Robert F Apo-2 ligand variants and uses thereof
US8076292B2 (en) 2001-10-10 2011-12-13 Novo Nordisk A/S Factor VIII: remodeling and glycoconjugation of factor VIII
US8716239B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
US8716240B2 (en) 2001-10-10 2014-05-06 Novo Nordisk A/S Erythropoietin: remodeling and glycoconjugation of erythropoietin
US20080187955A1 (en) * 2001-10-10 2008-08-07 Neose Technologies, Inc. Erythropoietin: remodeling and glycoconjugation of erythropoietin
US8008252B2 (en) 2001-10-10 2011-08-30 Novo Nordisk A/S Factor VII: remodeling and glycoconjugation of Factor VII
US20100261872A1 (en) * 2001-10-10 2010-10-14 Neose Technologies, Inc. Factor VIII: remodeling and glycoconjugation of factor VIII
WO2003040695A2 (en) * 2001-11-08 2003-05-15 Moyle William R Protein knobs
WO2003040695A3 (en) * 2001-11-08 2004-11-11 William R Moyle Protein knobs
US7557195B2 (en) 2002-03-20 2009-07-07 Biopolymed, Inc. Stoichiometric conjugates of biocompatible polymers at the unpaired cysteine residue of the wild-type G-CSF
US20050143563A1 (en) * 2002-03-20 2005-06-30 Myung-Ok Park Preparation of g-csf stoichiometrically conjugated with biocompatible polymers at cystein residue
US8318704B2 (en) 2002-05-08 2012-11-27 Neuronova Ab Modulation of neural stem cells and neural progenitor cells
US20110189184A1 (en) * 2002-05-08 2011-08-04 Neuronova Ab Modulation of neural stem cells and neural progenitor cells
US20080213276A1 (en) * 2002-05-08 2008-09-04 Per Lindquist Modulation of neural stem cells and neural progenitor cells
US7811822B2 (en) 2002-05-08 2010-10-12 Neuronova Ab Modulation of neural stem cells and neural progenitor cells
US8053410B2 (en) 2002-06-21 2011-11-08 Novo Nordisk Health Care A/G Pegylated factor VII glycoforms
EP2500032A1 (en) 2002-06-24 2012-09-19 Genentech, Inc. APO-2 ligand/trail variants and uses thereof
US20060177894A1 (en) * 2002-11-08 2006-08-10 Moyle William R Protein knobs
US20090075873A1 (en) * 2002-11-21 2009-03-19 Antonio Cruz Gastrin compositions and formulations, and methods of use and preparation
US7803766B2 (en) 2002-11-21 2010-09-28 Warath Pharmaceuticals, Inc Gastrin compositions and formulations, and methods of use and preparation
US20040126361A1 (en) * 2002-12-26 2004-07-01 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency
US20040136952A1 (en) * 2002-12-26 2004-07-15 Mountain View Pharmaceuticals, Inc. Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
US20080058246A1 (en) * 2002-12-26 2008-03-06 Mountain View Pharmaceuticals, Inc. Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity
US9125880B2 (en) 2002-12-26 2015-09-08 Mountain View Pharmaceuticals, Inc. Polymer conjugates of interferon-beta with enhanced biological potency
US8106131B2 (en) 2002-12-31 2012-01-31 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
US7432330B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
US8835556B2 (en) 2002-12-31 2014-09-16 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
US20040204548A1 (en) * 2002-12-31 2004-10-14 Antoni Kozlowski Hydrolytically stable maleimide-terminated polymers
US20090012241A1 (en) * 2002-12-31 2009-01-08 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
EP2186525A2 (en) 2002-12-31 2010-05-19 SYGNIS Bioscience GmbH & Co KG G-CSF for use in the treatment of Parkinson's disease
US7432331B2 (en) 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
EP2311483A1 (en) 2002-12-31 2011-04-20 SYGNIS Bioscience GmbH & Co KG GCSF for use in treating a neurological disease with pathophysiological mechanisms involving ischemia or hypoxia
US8227555B2 (en) 2002-12-31 2012-07-24 Nektar Therapeutics Hydrolytically stable maleimide-terminated polymers
US8791070B2 (en) 2003-04-09 2014-07-29 Novo Nordisk A/S Glycopegylated factor IX
US8853161B2 (en) 2003-04-09 2014-10-07 Novo Nordisk A/S Glycopegylation methods and proteins/peptides produced by the methods
US20040225097A1 (en) * 2003-05-07 2004-11-11 Kwang Nho Novel preparation method of peg-maleimide derivatives
US6828401B2 (en) * 2003-05-07 2004-12-07 Sunbio Inc. Preparation method of peg-maleimide derivatives
US20050163782A1 (en) * 2003-06-27 2005-07-28 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US20090041758A1 (en) * 2003-06-27 2009-02-12 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US7700097B2 (en) 2003-06-27 2010-04-20 Biogen Idec Ma Inc. Purification and preferential synthesis of binding molecules
US8603473B2 (en) 2003-06-27 2013-12-10 Biogen Idec Ma Inc. Modified binding molecules comprising connecting peptides
US20050163783A1 (en) * 2003-06-27 2005-07-28 Biogen Idec Ma Inc. Purification and preferential synthesis of binding molecules
US9005625B2 (en) 2003-07-25 2015-04-14 Novo Nordisk A/S Antibody toxin conjugates
US7771755B2 (en) 2003-09-12 2010-08-10 Wyeth Injectable calcium phosphate solid rods and pastes for delivery of osteogenic proteins
US20050090520A1 (en) * 2003-09-12 2005-04-28 Per Lindquist Treatment of disease or injury of the nervous system with FTY720
US8507008B2 (en) 2003-09-12 2013-08-13 Etex Corporation Injectable calcium phosphate solid rods and pastes for delivery of osteogenic proteins
US8916360B2 (en) 2003-11-24 2014-12-23 Novo Nordisk A/S Glycopegylated erythropoietin
US8653286B2 (en) 2003-12-03 2014-02-18 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
US7790835B2 (en) 2003-12-03 2010-09-07 Nektar Therapeutics Method of preparing maleimide functionalized polymers
US8895759B2 (en) 2003-12-03 2014-11-25 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
US8258324B2 (en) 2003-12-03 2012-09-04 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
US8632770B2 (en) 2003-12-03 2014-01-21 Novo Nordisk A/S Glycopegylated factor IX
US20100330060A1 (en) * 2003-12-03 2010-12-30 Novo Nordisk A/S Glycopegylated factor ix
US20050176922A1 (en) * 2003-12-03 2005-08-11 Mcmanus Samuel P. Methods of preparing maleimide functionalized polymers
US8039579B2 (en) 2003-12-03 2011-10-18 Nektar Therapeutics Intermediates useful in the preparation of maleimide functionalized polymers
US20070166278A1 (en) * 2004-04-15 2007-07-19 Veronese Francesco M Novel g-csf conjugates
US20060029573A1 (en) * 2004-06-30 2006-02-09 Chun Shen Pegylated interferon alpha-1b
EP2295069A1 (en) 2004-06-30 2011-03-16 Sygnis Bioscience GmbH & Co. KG Treatment of neurological disorders with haematopoietic growth factors
US7511016B2 (en) * 2004-07-07 2009-03-31 Mosamedix B.V. Annexins, derivatives thereof, and annexin-cys variants, as well as therapeutic and diagnostic uses thereof
US20060009381A1 (en) * 2004-07-07 2006-01-12 I.P. Randywyck B.V. Annexins, derivatives thereof, and annexin-cys variants, as well as therapeutic and diagnostic uses thereof
US8791066B2 (en) 2004-07-13 2014-07-29 Novo Nordisk A/S Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1]
US20070048855A1 (en) * 2004-09-17 2007-03-01 Alejandra Gamez Variants and chemically-modified variants of phenylalanine ammonia-lyase
US7553653B2 (en) 2004-09-17 2009-06-30 Biomarin Pharmaceutical Inc. Variants and chemically-modified variants of phenylalanine ammonia-lyase
US9200049B2 (en) 2004-10-29 2015-12-01 Novo Nordisk A/S Remodeling and glycopegylation of fibroblast growth factor (FGF)
US20090162380A1 (en) * 2005-01-05 2009-06-25 Scott Glaser Multispecific binding molecules comprising connecting peptides
US8084026B2 (en) 2005-01-05 2011-12-27 Biogen Idec Ma Inc. Multispecific binding molecules comprising connecting peptides
US9029331B2 (en) 2005-01-10 2015-05-12 Novo Nordisk A/S Glycopegylated granulocyte colony stimulating factor
US9187546B2 (en) 2005-04-08 2015-11-17 Novo Nordisk A/S Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
US7833979B2 (en) 2005-04-22 2010-11-16 Amgen Inc. Toxin peptide therapeutic agents
US8907071B2 (en) 2005-04-22 2014-12-09 Amgen Inc. Toxin peptide therapeutic agents
US20070071764A1 (en) * 2005-04-22 2007-03-29 Sullivan John K Toxin peptide therapeutic agents
US20080255026A1 (en) * 2005-05-25 2008-10-16 Glycopegylated Factor 1X Glycopegylated Factor Ix
US8404809B2 (en) 2005-05-25 2013-03-26 Novo Nordisk A/S Glycopegylated factor IX
US20100081791A1 (en) * 2005-05-25 2010-04-01 Novo Nordisk A/S Glycopegylated factor ix
US8088899B2 (en) 2005-06-13 2012-01-03 Cj Cheiljedang Corporation Human granulocyte-colony stimulating factor isoforms
US20100143288A1 (en) * 2005-06-13 2010-06-10 Cj Cheiljedang Corporation Human granulocyte-colony stimulating factor isoforms
US8633300B2 (en) 2005-06-17 2014-01-21 Novo Nordisk Healthcare Ag Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine
US20080200651A1 (en) * 2005-06-17 2008-08-21 Novo Nordisk Healthcare A/G Selective Reduction and Derivatization of Engineered Proteins Comprising at Least One Non-Native Cysteine
US20070049688A1 (en) * 2005-07-19 2007-03-01 Antoni Kozlowski Method for preparing polymer maleimides
US20110046315A1 (en) * 2005-07-19 2011-02-24 Nektar Therapeutics Method for Preparing Conjugates Using Polymer Maleimides
US8058385B2 (en) 2005-07-19 2011-11-15 Nektar Therapeutics Method for preparing conjugates using polymer maleimides
US8227558B2 (en) 2005-07-19 2012-07-24 Nektar Therapeutics Method for preparing polymer maleimides
US7872082B2 (en) 2005-07-19 2011-01-18 Nektar Therapeutics Method for preparing polymer maleimides
US20070092482A1 (en) * 2005-08-04 2007-04-26 Bossard Mary J Conjugates of a G-CSF moiety and a polymer
US8008453B2 (en) 2005-08-12 2011-08-30 Amgen Inc. Modified Fc molecules
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
EP2425860A1 (en) 2005-08-12 2012-03-07 Amgen Inc. Modified Fc molecules
US20090305967A1 (en) * 2005-08-19 2009-12-10 Novo Nordisk A/S Glycopegylated factor vii and factor viia
US8911967B2 (en) 2005-08-19 2014-12-16 Novo Nordisk A/S One pot desialylation and glycopegylation of therapeutic peptides
US20090163411A1 (en) * 2005-10-03 2009-06-25 Bolder Biotechnology, Inc. Long acting vegf inhibitors and methods of use
US8329866B2 (en) 2005-10-03 2012-12-11 Bolder Biotechnology, Inc. Long acting VEGF inhibitors and methods of use
US20070160658A1 (en) * 2005-10-20 2007-07-12 The Penn State Research Foundation Delivery system for diagnostic and therapeutic agents
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates
US8841439B2 (en) 2005-11-03 2014-09-23 Novo Nordisk A/S Nucleotide sugar purification using membranes
US20080008695A1 (en) * 2006-06-12 2008-01-10 Vellard Michel C Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
US7531341B1 (en) 2006-06-12 2009-05-12 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
US7534595B2 (en) 2006-06-12 2009-05-19 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
US8969532B2 (en) 2006-10-03 2015-03-03 Novo Nordisk A/S Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
US20090281028A1 (en) * 2006-10-25 2009-11-12 Sullivan John K OSK1 peptide analogs and pharmaceutical compositions
US7834164B2 (en) 2006-10-25 2010-11-16 Amgen Inc. DNA encoding OSK1 toxin peptide analogs and vectors and cells for combinant expression
US7820623B2 (en) 2006-10-25 2010-10-26 Amgen Inc. Conjugated toxin peptide therapeutic agents
US7803769B2 (en) 2006-10-25 2010-09-28 Amgen Inc. OSK1 peptide analogs and pharmaceutical compositions
US7910102B2 (en) 2006-10-25 2011-03-22 Amgen Inc. Methods of using conjugated toxin peptide therapeutic agents
US20090305399A1 (en) * 2006-10-25 2009-12-10 Sullivan John K DNA encoding OSK1 toxin peptide analogs and vectors and cells for combinant expression
US7825093B2 (en) 2006-10-25 2010-11-02 Amgen Inc. Methods of using OSK1 peptide analogs
US20090299044A1 (en) * 2006-10-25 2009-12-03 Sullivan John K DNA encoding chimeric toxin peptide fusion proteins and vectors and mammalian cells for recombinant expression
US8043829B2 (en) 2006-10-25 2011-10-25 Amgen Inc. DNA encoding chimeric toxin peptide fusion proteins and vectors and mammalian cells for recombinant expression
US9050304B2 (en) 2007-04-03 2015-06-09 Ratiopharm Gmbh Methods of treatment using glycopegylated G-CSF
EP2738257A1 (en) 2007-05-22 2014-06-04 Amgen Inc. Compositions and methods for producing bioactive fusion proteins
US8420779B2 (en) 2007-05-22 2013-04-16 Amgen Inc. Compositions and methods for producing bioactive fusion proteins
US9493499B2 (en) 2007-06-12 2016-11-15 Novo Nordisk A/S Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
US7537923B2 (en) 2007-08-17 2009-05-26 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
US20090047268A1 (en) * 2007-08-17 2009-02-19 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
US7790433B2 (en) 2007-08-17 2010-09-07 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
US7560263B2 (en) 2007-08-17 2009-07-14 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
US20100278802A1 (en) * 2007-08-17 2010-11-04 Biomarin Pharmaceutical Inc. Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
US20090263369A1 (en) * 2007-08-17 2009-10-22 Biomarin Pharmaceutical Inc. Compositions of Prokaryotic Phenylalanine Ammonia-Lyase and Methods of Treating Cancer Using Compositions Thereof
EP2195338A4 (en) * 2007-10-01 2010-11-03 Pharmaessentia Corp N-terminal modified interferon-alpha
EP2195338A1 (en) * 2007-10-01 2010-06-16 Pharmaessentia Corp. N-terminal modified interferon-alpha
US8106160B2 (en) 2007-10-01 2012-01-31 Pharmaessentia Corp. N-terminal modified interferon-alpha
US20090269306A1 (en) * 2007-10-01 2009-10-29 Pharmaessentia Corp. N-terminal modified interferon-alpha
WO2009046080A1 (en) 2007-10-01 2009-04-09 Pharmaessentia Corp. N-terminal modified interferon-alpha
CN101883784B (en) 2007-10-01 2014-10-15 药华医药股份有限公司 N- terminally modified interferon -α
US9150848B2 (en) 2008-02-27 2015-10-06 Novo Nordisk A/S Conjugated factor VIII molecules
US20110201022A1 (en) * 2008-07-30 2011-08-18 Biomarin Pharmaceutical Inc. Assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase
US9557340B2 (en) 2008-07-30 2017-01-31 Biomarin Pharmaceutical Inc. Assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase
WO2012125973A2 (en) 2011-03-16 2012-09-20 Amgen Inc. Potent and selective inhibitors of nav1.3 and nav1.7
WO2014099984A1 (en) 2012-12-20 2014-06-26 Amgen Inc. Apj receptor agonists and uses thereof
WO2014165277A2 (en) 2013-03-12 2014-10-09 Amgen Inc. POTENT AND SELECTIVE INHIBITORS OF Nav1.7
WO2015191781A2 (en) 2014-06-10 2015-12-17 Amgen Inc. Apelin polypeptides
WO2017075173A2 (en) 2015-10-30 2017-05-04 Genentech, Inc. Anti-factor d antibodies and conjugates

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